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1 ADENO-ASSOCIATED VIRUS (AAV)-MEDIATED JOINT RESTORATION APPROACHES: CHARACTERIZATION AND APPLICATION by Hannah Hoeun Lee BS, Cornell University, 2006 Submitted to the Graduate Faculty of Swanson School of Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2012

2 UNIVERSITY OF PITTSBURGH SWANSON SCHOOL OF ENGINEERING This dissertation was presented by Hannah Hoeun Lee It was defended on May 22, 2012 and approved by Partha Roy, PhD, Associate Professor, Departments of Bioengineering and Pathology Yadong Wang, PhD, Associate Professor, Department of Bioengineering Xiao Xiao, PhD, Professor, Division of Molecular Pharmaceutics, University of North Carolina Dissertation Director: Constance Chu, MD, Professor, Departments of Orthopaedic Surgery and Bioengineering ii

3 Copyright by Hannah Hoeun Lee 2012 iii

4 ADENO-ASSOCIATED VIRUS (AAV)-MEDIATED JOINT RESTORATION APPROACHES: CHARACTERIZATION AND APPLICATION Hannah Hoeun Lee, PhD University of Pittsburgh, 2012 Osteoarthritis is the leading cause of morbidity world-wide. Articular cartilage has limited regenerative potential and effective disease-modifying treatments for osteoarthritis are lacking. Hence, tissue engineering strategies to enhance cartilage repair potential are widely investigated. Adeno-associated virus (AAV) is a promising gene vector for articular cartilage and has already been used in clinical trials. We hypothesized that gene delivery of bioactive molecules to articular joints using AAV is efficacious to promote joint restoration. In the first two parts of the study, we showed persistent, localized, and controllable transgene expression within intact and injured joints following a single intra-articular injection of AAV. Most of the transduction occurred in the intra-articular soft tissues for intact joints and when the AAV was injected prior to joint injury, whereas some cartilage transduction but limited soft tissue transduction was seen when the AAV was injected following joint injury. Doxycycline was used as an induction agent of the AAV-mediated transgene expression. In the third part of the study, doxycycline was shown to have matrix-metalloproteinase-13 suppressing function during mesenchymal stem cell (MSC) chondrogenesis as well as in vivo cartilage repair. This supports potential use of doxycycline either alone or in gene therapy applications to improve cartilage repair. In the fourth part of the study, we optimized native biomaterial fibrin glue (FG) for its function as an AAVreleasing scaffold for tissue engineering applications. Bioactive AAV from diluted FG showed iv

5 improved release, transduction efficiency, and chondrogenic effect on MSC. Lastly, AAVs encoding for anabolic and/or anti-inflammatory factors were explored to enhance MSC chondrogenesis in an inflammatory milieu. The MSCs transduced with AAV-interleukin-1 receptor antagonist (IRAP), but not AAV-transforming growth factor (TGF)-β 1, resulted in improved chondrogenic potential. In conclusion, various aspects of AAV-mediated joint restoration approaches were investigated, from characterization of AAV delivery by direct injection or with the use of FG, to therapeutic applications with doxycycline, AAV-IRAP, and AAV-TGF-β 1. These results support continued investigations of therapeutic potential of AAV for safe, localized, and controlled delivery of bioactive substances to promote joint restoration, which may delay/prevent the onset of debilitating osteoarthritis. v

7 1.4.3 Objective #3: To test the hypothesis that fibrin glue concentration alters the adeno-associated virus-release profile, which affects adeno-associated virus-bioavailability Objective #4: To test the hypothesis that chondrogenic potential of human mesenchymal stem cells will be enhanced by using adeno-associated virus encoding for anabolic factors and/or anti-catabolic factors SINGLE INTRA-ARTICULAR INJECTION OF ADENO-ASSOCIATED VIRUS RESULTS IN STABLE AND CONTROLLABLE IN VIVO TRANSGENE EXPRESSION IN NORMAL RAT KNEES INTRODUCTION METHODS AAV vector production In vitro infection of rat articular chondrocytes Animal experiments Statistical analysis RESULTS In vitro response of rat articular chondrocytes to AAV2 vectors Duration of in vivo transgene expression after a single intra-articular injection of AAV Localization of in vivo transgene expression after a single intra-articular injection of AAV Controlled in vivo transgene expression following a single intra-articular injection of AAV DISCUSSION CONCLUSIONS PERSISTENCE, LOCALIZATION, AND EXTERNAL CONTROL OF TRANSGENE EXPRESSION AFTER SINGLE INJECTION OF ADENO- ASSOCIATED VIRUS INTO INJURED JOINTS INTRODUCTION vii

17 PREFACE First and foremost, I thank God for the incredible opportunities he has presented for me. It is by your grace that I am here today, and I dedicate this to you. I would like to express my deepest appreciation to my dissertation advisor, Dr. Constance Chu. Thank you for providing me with the opportunity to be a member of your wonderful team. It has been a very rewarding experience and I truly appreciate the constant support and guidance you have provided. It was very helpful to have a clinician-scientist mentor that has such a great enthusiasm for research, and thus is a great role-model for my MD/PhD training. To Drs. Roy, Wang, and Xiao: thank you for serving on my dissertation committee. I truly appreciate your time, advice, and guidance. To Drs. Clayton Wiley, Richard Steinman, and George Stetten; thank you for your endless support from the Medical Scientist Training Program (MSTP) side. A special thank you to Dr. Harvey Borovetz. You have challenged and encouraged me about my career path from the interview day and throughout all these years, and I appreciate all your help. Thank you also to CATER (Cellular Approaches in Tissue Engineering and Regeneration) faculty members: Drs. Alan Russell, Paul Monga, and Kacey Marra. I would like to thank all the members, present and past, of the Cartilage Restoration Center for their help and kindness. To Karin Payne, who is a mentor and a friend, you have truly guided my development as a scientist and I appreciate all your help. Thank you to Christian xvii

18 Coyle, Amgad Haleem, Veronica Yao, and Jean Zamzow, who have been incredibly helpful. I would also like to thank Kimberly Suter, Michele Mulkeen, and Cecilia Huang, who has provided me with technical assistance on my projects. Thank you to all the Orthopaedic Surgery Residents who spent their research years in Cartilage Restoration Center, doing projects that are/are not related my work and have provided guidance in clinical realms: Venkat Seshadri, David Bear, Sarah Henry, Michael O Malley, and Nicole Friel. In addition, I would like to acknowledge other members of the Chu lab: Ashley Williams, Megan Bowers, Kimberly Diamond, Steve Bruno, Patricia Stepp, and Andrew Lynch. Endless thank you to all the students in the MSTP for their friendship and scientific camaraderie. Special call out to Jeffrey Wong, Lolita Nidadavolu, and John Kang, who started the MSTP training with me. As well, thank you to Ken Urish and Joe Vella for their guidance through Bioengineering program as an MD/PhD student. Thank you for my fellow School of Medicine and Bioengineering students. I learned everyday from you and it was a pleasure working with all of you. Deep gratitude to my KCCP family, for their prayers and support. I would especially like to note Judong Lee and Junchol Park for their help in technical areas. I really appreciate it! Most of all, thank you to my parents and Debbie. I love you all so much and you are the reason that I am who I am today. I can t possibly express enough gratitude! Lastly, I would like to thank the National Institutes of Health (NIH) and the National Institute of Biomedical Imaging and Bioengineering for providing me with the T32 Institutional National Research Service Award through the CATER program for generously providing me with funding for the last two years of graduate school. This work was also supported by funding to Dr. Constance Chu from the NIH, Chondrogenesis in situ (R01 AR051963), Multicenter xviii

20 1.0 INTRODUCTION: OSTEOARTHRITIS Osteoarthritis (OA) is the most common type of arthritis in the world and the single largest cause of disability (1, 2). According to the World Health Organization (WHO), OA is among the top 10 conditions representing a global disease burden (3). It affects nearly 27 million people and costs almost $128 billion per year in medical care and indirect expenses in the US alone (4, 5). OA is symptomatically characterized by joint pain, tenderness, occasional effusions, deformity, and eventually loss of joint function with restrictions of motion (6). Clinical signs of OA include progressive degeneration of articular cartilage, which leads to fibrillation, ulceration, and joint space narrowing. Pathophysiology of OA is multi-factorial, but believed to be a combination of genetic, mechanical, and biochemical factors (7). It is traditionally regarded to result from wear and tear of the joint and hence associated with aging and obesity (8, 9). However, concerns with subjects with high physical activity, such as high impact sports athletes, and their risk of developing OA are also high (10, 11). Many studies have found a link between the development of OA and a history of participation in sports at an elite or professional level, especially for soccer, hockey, American football, and tennis (12). 1

21 1.1 ARTICULAR CARTILAGE JOINT Articular cartilage joint in physiology The articular surface plays an essential role of allowing smooth transmission of force between two bones, thereby permitting painless movements of the joint (13). Articular cartilage is an avascular, aneural, and alymphatic tissue with a sparse population of chondrocytes embedded within a highly organized, dense extracellular matrix (ECM) (14). The mesenchymal cell-derived chondrocytes are the single cell type within the cartilage, and they maintain the homeostasis of the extensive framework of ECM via the synthesis of various anabolic and catabolic factors. The ECM is primarily composed of water, collagen type II (col-ii), and aggrecan, which is a large aggregating proteoglycan that is highly hydrated due to the negative charge density of its sulfated glycosaminoglycan (GAG) (15). The col-ii provides tensile strength while aggrecan allows the cartilage to resist compressive load and provides a friction-free articulating surface. The catabolic factors involved in cartilage homeostasis include matrix metalloproteinases (MMPs) that degrade collagens, and aggrecanases, such as a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTSs), which degrade aggrecans (14). With these compositions, the articular cartilage is a fluid-rich structure that functions in load-bearing via controlling the fluid movement in order to disperse the load/weight throughout the cartilage and to the underlying bone. As well, joint movement facilitates synovial fluid circulation and nutrient delivery to cartilage. However, due to the limited intrinsic capacity for self-repair, articular cartilage injuries due to trauma or degenerative inevitably progress to OA (16). In addition to articular cartilage, there are number of soft-tissue structures that are integral components of the joint. The anterior cruciate ligament (ACL) and menisci are the two 2

22 most relevant structures for the knee joint, as they are essential for joint stability and are commonly injured in the general population as well as in athletes (17). ACL connects the femur to tibia and is essential for the normal motion and stability of knee joints (18). It mainly consists of fibroblasts and ECM composed of non-parallel collagen types I and III fibers with multiple fascicles and is surrounded by synovium (18-20). ACL also contains small amounts of proteoglycans and glycoproteins (20). There are two bundles to the ACL, anteromedial and posterolateral, respective of their position on the tibia, and limits the forward motion of the tibia (21). ACL, alike articular cartilage, has a limited capacity to heal and regenerate after injury due to poor vascularity (22). Menisci are a pair of wedge-shaped semilunar fibrocartilages that are interposed between the femoral condyles and tibial plateaus of the knee joint (23). They function to correct the incongruency between these two articular cartilage surfaces, increasing the contact area in the femorotibial joint and reducing the stresses on tibial cartilage. Additional roles serves by menisci are shock absorption, stability enhancement, and lubrication (24). Molecular compositions of the menisci are mainly collagen type I (col-i), and small portions of collagens type II, III, V, and VI (25). Menisci also contain proteoglycan at a lesser degree than hyaline cartilage Articular cartilage joint in pathology OA involves all joint components: cartilage, synovial membrane/synovium, and subchondral bone. During the OA pathogenesis, slow focal destruction of articular cartilage leads to a roughening and thinning of the weight-bearing regions of the articular surface, resulting in progressive immobility and pain (26). The process begins with the disruption of the cartilage cell-matrix interactions, with the alteration of matrix molecular structure and composition and 3

23 the loss of aggrecans. Inflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), and synovial inflammation have also been implicated as important mediators of the OA progression. These inflammatory cytokines upregulate expressions of MMPs and ADAMTSs (14). They also stimulate the chondrocytes to proliferate and synthesis more matrix components in an attempt to heal the articular cartilage. However, the chondrocytes proliferative potential and ECM synthesis capacity declines with time and age (27). The synovium is also capable of accelerating the articular cartilage degradation by degenerating soluble aggrecanases and inflammatory cytokines (15, 28). The col-ii and aggrecan breakdown products have been shown to accumulate in the cartilage ECM as well as the synovial fluid during the OA process. Therefore, with progressive joint degeneration, the anabolic response eventually becomes overwhelmed by the catabolic activities, leading to progressive thinning of articular cartilage and to OA (14). Indirect cartilage injury and subsequent arthritis development due to joint instability induced by damaging the extra-cartilage soft tissue structure is also a common pathological process for the knee joint. Both ACL and menisci injuries have been shown to be followed by arthritic changes of the knees (29). In addition to the initial high force that damages intraarticular structures involved in the trauma, the ACL and menisci ruptures initiate a cascade of pathogenic processes in the acute phase that can lead to the development of OA in itself (17, 30). In addition to acute effects, the lack of a functionally normal ACL or menisci leads to chronic changes in the static and dynamic loading of the knee and increase forces on the cartilage and other joint structures. Consequently, additional damages commonly occur in the ACL and/or menisci-injured knees, further contributing to initiation and progression of OA development (31, 32). 4

24 1.2 CURRENT TREATMENTS AND LIMITATIONS Clinical strategies for management of cartilage pathology have evolved from primarily palliative methods to treat early disease until joint replacement for end-stage disease, toward more biologic, joint-conserving measures. These include cartilage reconstruction as in osteochondral graft transplantation (33), and cartilage repair as in bone marrow (BM) stimulation (34, 35), autologous chondrocyte implantation (36), or stem cell implantation (37). However, results of cartilage repair procedures have been equivocal, and no single approach has been shown to be superior (38, 39). Therefore, an important strategy for cartilage repair and tissue engineering is on investigating utilization of bioactive factors and biodegradable scaffolds to promote local, recruited, or transplanted cell proliferation and differentiation within cartilage wounds. Despite the promising results of in vitro growth factor application, in vivo application has not been as successful due to the short half-life of these growth factors and the need for high repetitive dosing as well as a costly purification process. 1.3 TISSUE ENGINEERING AND GENE THERAPY FOR ARTICULAR CARTILAGE RESTORATION Adeno-associated virus and gene therapy Gene therapy can overcome the obstacles of short half-life, need for high repetitive dosing, and costly purification process associated with growth factor applications by delivery genes that encode chondrogenic growth factors or inhibitors of cartilage catabolites in various vector 5

25 constructs. This also provides for a local delivery system that drives the expression of therapeutic molecules over an extended period and overcome the need for repeated administrations or interventions. Gene carriers include plasmids, non-viral vectors, and viral vectors. Although plasmids and non-viral vectors are less toxic, less immunogenic, and easier to prepare, their gene delivery efficiencies are significantly lower compared to viral vectors (40). Hence, viral vectors are currently considered the most effective agents for in vivo gene transfer (41). Viral vectors that have been used in the pre-clinical setting include adenovirus, adenoassociated virus (AAV) (42), and lentivirus (43). Many delivery vehicles can successfully target synovium and other soft tissues surrounding the articular cartilage (44-46). However, cartilage is particularly challenging for any type of transportation due to its highly impermeable and negatively charged ECM. AAV is derived from an endemic, non-pathogenic parvovirus. It has the following advantages over other viruses: sustained transgene expression over longer period, reduced potential for host immune response, and the capacity to transduce both dividing and non-dividing cells (47). After transduction, the viral genome translocates into the target cell nucleus, and the DNA polymerase generates the transducing episome. Numerous serotypes of AAV have been identified, each having different preferential targets. Among the different serotypes, AAV serotype 2 (AAV2) is considered to have the best defined safety profile as it has already been used in clinical trials (48). There are three general approaches for cartilage viral gene therapy applications: direct, indirect, and hybrid. The direct method involves the injection of viral vectors directly into the articular joint space to transducer local cells (47). The indirect approach involves the use of genetically modified cells (42), and/or biodegradable scaffolds that release genetically modified 6

26 cells (49, 50). This leads to the site-specific release of bioactive molecules important in tissue regeneration, though temporary. In addition, this approach requires ex vivo manipulation of cultured cells, which faces high barriers with respect to clinical translation. The third hybrid approach involves implantation of biodegradable scaffolds embedded with genetic materials (51, 52), leading to direct and localized in situ delivery of bioactive vectors to the host cells in proximity to the scaffold, as well as the sustained expression of the transgene Scaffold Biomaterial: Fibrin Glue Fibrin glue (FG) is a natural substance available for human use clinically as a commercial product or readily prepared from plasma for autologous applications (53). These properties mean that laboratory findings may be more readily translated into potential human clinical applications than similar findings with synthetic polymers. Despite being widely used in clinical surgical practice as an adhesive and sealant for hemostasis (54), only in recent years has the ability of FG to act as a delivery scaffold received attention (55). Besides being commercially available for sterile applications, it is non-immunogenic, biodegradable, and malleable so that it can be molded into irregular tissue defects. All of these advantages make FG an appealing scaffold for pre-clinical translational cartilage tissue engineering applications Cell types used in joint restoration Tissue engineering strategies for cartilage repair with autologous cell sources mostly involves use of chondrocytes isolate from cartilage (56), or mesenchymal stem cells (MSCs) from bone marrow (57), fat (58), and muscle (59). While autologous chondrocytes has good 7

28 Treatment strategies based on the use of bone-marrow derived cartilage progenitor stem cells, such as MSCs, and growth factors have been extensively studied using well-established in vitro chondrogenic assays and in vivo animal models (61). Transforming growth factor-β (TGF-β) is the most extensively examined anabolic growth factor for hmsc induction into chondrogenic phenotype in vitro (57, 62), especially the TGF-β 1 isoform (63). Relevant TGF-β 1 signaling pathway (Fig. 1-1) include TGF-β 1 binding to its type II receptor (TGF-β-R-II), which then forms a complex with the type I TGF-β receptor (64). Upon the receptor activation, intracellular signaling molecule Smad2 or Smad3 gets phosphorylated, forms a complex with Smad4, and the complex then translocates to the nucleus to function as a transcription factor. Via this mechanism, TGF-β 1 stimulates collagen-ii and proteoglycan expressions in MSCs (65). Smad7 inhibits Smad2 and Smad3 phosphorylation and prevents further signaling. In addition to its anabolic functions, TGF-β 1 also decreases the catabolic activity of inflammatory cytokines, such as IL-1, in vitro and in vivo (64). However, a number of deleterious side effects have been associated with uncontrolled high-dose TGF-β 1 administration in vivo, including synovial fibrosis and osteophyte formation (61, 66, 67). These side effects have been shown to be blocked when Smad7 was intracellularly overexpressed in the synovial lining while maintaining TGF-β 1 induced cartilage repair effects (68). On the catabolic side, IL-1β and TNF-α are pro-inflammatory cytokines that activate the nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) pathway, induce destructive processes in multiple cell types in the joint space, and thus play a critical role in the progression of OA (26, 69). These cytokines accelerate the switching of the anabolic to catabolic states of the cartilage, and it has been shown that each of their stimulation increases the expression of catabolic enzymes, such as ADAMTS-5 (69, 70) and MMP-13 (71). As well, IL- 9

29 1β and TNF-α have been shown to inhibit TGF-β 1 -mediated hmsc chondrogenesis (71). Previous animal studies showed that TNF is a potent pro-inflammatory mediator and IL-1 a potent cartilage catabolic mediator (72). Arthritis induced by TNF-overexpression was completely blocked by antibodies targeting IL-1 receptor, suggesting that IL-1 is the pivotal downstream mediator in arthritis. IL-1 receptor antagonist (IL-1Ra) is a small kda secreted protein that competitively inhibits IL-1 by binding to and preoccupying type I IL-1 receptors, thus preventing IL-1 ligand-receptor interactions (73). It has no known agonist activity (74). However, although this strategy is straightforward, the efficacy is limited by the poor pharmacokinetics of the small molecule and the need for high dosing to fully occupy IL-1 receptors for prolonged periods of time. 1.4 PROJECT OBJECTIVES The central hypothesis of this project is to test the hypothesis that modulation of various anabolic and catabolic factors involved in mesenchymal stem cell (MSC) chondrogenesis via adenoassociated virus (AAV) will enhance the outcomes of joint restoration Objective #1: To test the hypothesis that the transgene expression in articular cartilage joint is localized, persistent, and controllable following a single intraarticular injection of adeno-associated virus Adeno-associated virus (AAV) is a promising delivery vehicle for articular cartilage. Most simple method to deliver the genetic material for cartilage viral gene therapy applications is by 10

30 direct injection for the resident cells within the joint to produce the therapeutic factor. Knowledge of the duration of transgene expression is critically important to develop efficient therapies. As well, while long-term transgene expression may be critical for disease treatment, it may not be ideal for cartilage repair strategies, where a growth factor may only be needed for a limited time period. In cases where a growth factor is needed for a specific period of time, it may be advantageous to use an inducible gene expression system, where the transgene expression can be controlled pharmacologically by oral administration of the inducing agent. The tetracyclineinducible gene regulation system has been efficient both in vitro and in vivo (75-77). Doxycycline, an analog of tetracycline, is widely accepted as an inducer due to its safe use in humans (78). In this objective, AAV-mediated transgene persistence, duration, and controllability will be studied in intact rat diarthrodial joint using a reporter gene luciferase. Also, since the intact and undamaged articular cartilage is unlikely to require treatment, AAV transduction and transgene expression profiles in rat models of articular joint damage will provide invaluable information to improve gene therapy strategies, as they will more closely mimic the disease environment. Two different AAV injection time points, pre- and post-injury, will be investigated on osteochondral defect and anterior cruciate ligament transection models of joint damage Objective #2: To test the hypothesis that doxycycline reduces MMP, improves chondrogenesis of human bone marrow derived mesenchymal stem cells (MSC), and does not adversely affect repair of osteochondral defect (OCD) New strategies to improve repair tissue quality after bone marrow cell based procedures such as microfracture may reduce later development of osteoarthritis (OA). Doxycycline can be used to 11

31 control intra-articular transgene expression and has been shown to reduce matrix metalloproteinases (MMP) and OA disease progression. In this study, MSCs will be cultured in chondrogenic media with either 0, 1 or 2-μg/mL doxycycline to assess the doxycycline effect on chondrogenic differentiation. Rats will be treated with or without oral doxycycline following osteochondral defect surgeries to assess the doxycycline effect on cartilage repair. These studies will evaluate the potential use of doxycycline either alone or in combination with AAV gene therapy applications to improve cartilage repair and delay OA Objective #3: To test the hypothesis that fibrin glue concentration alters the adeno-associated virus-release profile, which affects adeno-associated virusbioavailability Use of biodegradable scaffolds embedded with AAV can lead to direct and localized in situ delivery of bioactive vectors to the host cells in close proximity to the scaffold. Although numerous novel synthetic and natural polymers have been investigated for cartilage tissue engineering studies, usage of an FDA-approved and autologous biomaterial as an AAV-releasing scaffold can accelerate the transition of basic science research into human clinical studies. Fibrin glue (FG) is a biomaterial widely used in clinical practice and tissue engineering applications. Nonetheless, FG formulation for AAV delivery has not been optimized. FG hydrogels containing varying fibrinogen concentrations will be characterized for its function as an AAV-releasing scaffold in vitro. Specifically, alterations in FG scaffold microstructure via different dilutions and its effect on AAV-release capacity will be characterized. As well, usage of biodegradable FG scaffold for release and delivery of bioactive AAVs to hmscs will be done by assaying for the induction of cartilage specific genes. 12

32 1.4.4 Objective #4: To test the hypothesis that chondrogenic potential of human mesenchymal stem cells will be enhanced by using adeno-associated virus encoding for anabolic factors and/or anti-catabolic factors Anabolic growth factors have been extensively studied for hmsc chondrogenesis and cartilage repair (16, 42, 57, 65, 79). However, the catabolic side of the balance, which is increasingly recognized to be important, warrants additional investigation. Interleukin-1 (IL-1) is the pivotal inflammatory cytokine in articular cartilage pathologies, with its levels greatly upregulated within the joint in disease processes (71). Furthermore, it has been shown the IL-1β inhibits hmsc chondrogenesis (71, 80). Therefore, anti-il-1 strategy like IL-1 receptor antagonist protein (IL-1Ra or IRAP), which competitively inhibits IL-1, holds further therapeutic potential (81). Transforming growth factor-β (TGF-β) is the most extensively examined anabolic growth factor for hmsc induction into chondrogenic phenotype and its use in chondrogenic media has long been established (61, 62). However, it has also been shown to blunt cytokine-mediated inhibition of hmsc chondrogenesis (71). As a basis to apply IRAP and TGF-β 1 antiinflammatory strategies in clinical MSC-mediated cartilage repair, AAV-IRAP transduced hmscs and AAV-TGF-β 1 transduced hmscs will be investigated for their chondrogenic differentiation in an inflammatory environment. 13

33 2.0 SINGLE INTRA-ARTICULAR INJECTION OF ADENO-ASSOCIATED VIRUS RESULTS IN STABLE AND CONTROLLABLE IN VIVO TRANSGENE EXPRESSION IN NORMAL RAT KNEES 2.1 INTRODUCTION Prevention of articular cartilage degradation or treatment of its damage in arthritis remains challenging due to the limited self-repair potential of articular cartilage. Currently, no therapeutic methods exist for complete re-establishment of cartilage function. Delivery of therapeutic agents that could promote articular cartilage repair or prevent its further degradation once damaged is an attractive therapeutic option. Protein biologics can be delivered systemically, or locally by direct injection or through polymer based delivery systems (82). However, due to the short half-life of proteins, administration of supra-physiological doses and/or repeated delivery are often necessary, significantly increasing the cost of these approaches. An attractive alternative is to deliver the genetic information to cells within the joints and engineer them to produce the therapeutic protein in situ. Naked DNA, retrovirus, adenovirus, and herpes virus-based vectors have been explored for gene transduction in vivo; however, most were rendered suboptimal due to safety, efficacy, and duration issues. Recombinant adeno-associated virus (AAV) derived from an endemic and non-pathogenic parvovirus is emerging as a promising delivery vehicle for musculoskeletal 14

34 tissues, with the advantages of sustained transgene expression, reduced potential for host immune response, and the capacity to transducer both dividing and non-dividing cells in vitro and in vivo (83). Different serotypes of AAV exist, each having different preferential targets (83, 84). AAV5 has better transduction efficacy in rodent arthritic joints; however, AAV2 is currently used in human clinical trials for arthritis (85-88). Many studies have genetically engineered cells ex vivo for implantation in vivo. Although this has led to cartilage repair in animal models (42, 89-92), culturing cells ex vivo prolongs the time before treatment and is also very expensive in a clinical setting. An alternative method would be direct injection and rely on the cells within the joint to produce the therapeutic factor. AAV vectors have a strong tropism for synoviocytes in vitro (93-96). However, few studies have identified the exact in vivo localization of the transgene, after intra-articular injection of AAV (44, 46, 79, 97-99). It is important to identify which cells or tissues are infected by AAV after intra-articular injection and to use this information to develop optimal treatment strategies for intra-articular disease processes such as inflammatory and degenerative arthritis. Knowledge of the duration of transgene expression is also important to develop efficient therapies. It has been reported that parkinsonian nonhuman primates having received AAV2 encoding human L-amino acid decarboxylase (L-DOPA) into their brains were still expressing the transgene after 8 years (100). AAV-mediated erythropoietin delivery to rhesus monkey skeletal muscle has led to transgene expression for more than 6 years (77). Long-term transgene expression may be critical for disease treatment, but may not be ideal for cartilage repair strategies, where a growth factor may only be advantageous to use an inducible gene expression system, where the transgene expression can be controlled pharmacologically by oral administration of the inducing agent. The tetracycline-inducible gene regulation system has been 15

35 efficient both in vitro and in vivo (75-77). Doxycycline (Dox), an analog of tetracycline, is widely accepted as an inducer due to its safe use in humans (78). Only few studies have investigated the use of an AAV-based tetracycline-inducible gene system in the knee joint after direct injection. Given the potential translational value of AAV for human clinical use, the aim of the present study was to investigate the presence, persistence, and ability to externally control transgene expression when using AAV2 delivered by a single intra-articular injection into the rat diarthrodial joint. The immunocompetent rat was chosen since it is a good animal model for future studies that will test the repair of articular cartilage, the prevention of its degradation, or the treatment of inflammatory arthritis by AAV-mediated gene therapy. 2.2 METHODS AAV vector production Double-stranded serotype 2 AAV (AAV2) vectors were produced using the three-plasmid cotransfection method (101). Depending on the AAV2 vector being produced, the first plasmid used was either the AAV-cytomegalovirus (CMV)-Luc plasmid with the luciferase (Luc) gene driven by the CMV promoter, the AAV-CMV-enhanced green fluorescent protein (GFP) plasmid, the AAV-CMV-reverse-tetracycline-controlled transactivator (rtta) plasmid, or the AAV-TRE- Luc plasmid with the Luc gene driven by the tetracycline response element (TRE) promoter. The second plasmid used in the cotransfection was the pxx6 plasmid, which contains the helper genes from adenovirus, and the third plasmid was the pxx2, which supplies AAV2 rep protein 16

36 and capsid protein (101). Vector purification was performed as previously described and AAV genomic titers were determined by DNA dot-blot assay (101) In vitro infection of rat articular chondrocytes Primary rat articular chondrocytes were extracted from the tibial and femoral cartilage of 3- month-old male Sprague Dawley rats. Cartilage samples were washed in phosphate buffered saline (PBS; Invitrogen) with 2% penicillin-streptomycin (pen/strep; Invitrogen). To digest the cartilage, thin shavings were incubated in 0.2% pronase (Calbiochem) solution in F-12 medium (Invitrogen) supplemented with 5% fetal bovine serum (FBS) and 1% pen/strep for 90 min in a dry 37 C incubator. This was followed by overnight digestion in 0.025% collagenase P (Roche Applied Science) in F-12 medium supplemented with 5% FBS and 1% pen/strep. Recovered chondrocytes were seeded as monolayers in a T-75 flask in 50% Dulbecco s Modified Eagle medium/50% F-12 medium (DMEM/F-12; Invitrogen) supplemented with 10% FBS, and 1% pen/strep under normal growth conditions (37 C, 5% CO 2 ). Cells were grown to passage four and plated onto 96-well plates at a density of 2.0 x 10 4 cells/cm 2 in growth medium (DMEM/F- 12, 10% FBS, 1% pen/strep). On the following day, chondrocytes in plain DMEM/F-12 were infected with 3.2 x 10 5 vector genomes per cell (vg/cell) of AAV-CMV-GFP, AAV-CMV-Luc, or AAV-CMV-rtTA + AAV-TRE-Luc for 1 h, after which the medium was changed to growth medium. This dose was selected because it gave the greatest gene expression in an in vitro dose response study ranging from 4 x x 10 5 vg/cell (data not shown). After 2 days, cells infected with AAV-TRE-Luc were stimulated with Dox at concentrations of 0, 1, or 2 μg/ml for 36 h. Luciferase transgene expression was measured 4 days post-transduction by adding 50 μl of luciferin (30 mg/ml; D-Luciferin Firefly, Caliper Life Sciences) per well and imaging after 5 17

37 minutes with the IVIS 200 optical imaging system (Xenogen Corp., Hopkinton, MA, USA). GFP signal was visualized on the same day using the epifluorescent Eclipse TE-2000U inverted microscopy (Nikon) Animal experiments All animal experiments were performed following a University of Pittsburgh Institutional Animal Care and Use Committee approved protocol. The study was divided into two parts. In the first study, nine male Sprague Dawley rats (3 months old) received a 50 μl intraarticular injection containing 2.5 x vg of AAV-CMV-GFP or AAV-CMV-Luc into the right and left knee joint, respectively. For longitudinal evaluation of luciferase expression, rats were anesthetized with isoflurane inhalation, followed by a 50 μl intra-articular injection of luciferin (30 mg/ml). After 5 min, to allow proper distribution of luciferin, rats were placed in an IVIS 200 optical imaging system. Photon emissions in the region of interest were quantified using Living Image software v.3.0 (Xenogen Corp.), and bioluminescent flux was reported as photons/sec. Rats were imaged every other week for 4 months, and monthly thereafter, until 1 year post-aav injection. Rats were sacrificed at either 1 week (N = 1), 2 weeks (N = 2), 3 months (N = 1), 4 months (N = 1), or 1 year (N = 4) after AAV injection. On the day of sacrifice, rats received an intraperitoneal injection of 100 μl of luciferin to determine whether any signal was present outside of the intra-articular space. They also received an intra-articular injection of luciferin and imaged as previously described. The joint was then opened to image the intraarticular tissues. Tissues within the joint were also analyzed for GFP-positive cells using a fluorescent stereomicroscope (MVX-10 MacroView Systems, Olympus, Japan) equipped with a 18

38 DP71 camera (Olympus). All GFP images in Fig. 2-4 had the contrast increased by the same value in Adobe Photoshop to improve the GFP signal for print. In the second animal experiment, the right and left knees of four male Sprague Dawley rats (3 months old) were divided into experimental and control groups. In the right knee, each rat received a 50 μl intra-articular injection containing 2.5 x vg of AAV-TRE-Luc and 2.5 x of AAV-CMV-rtTA. In the left knee, each rat received a 50 μl intra-articular injection containing 2.5 x vg of AAV-CMV-Luc and 2.5 x vg of AAV-CMV-rtTA, to have the same number of vg in both knees. To induce expression of luciferase in the right knee joint (injected with AAV-TRE-Luc), rats were administered drinking water containing Dox at a concentration of 2 mg/ml for 7 days, followed by its removal. To ensure fresh supply, the water was replaced every day and protected from light with amber drinking bottles. Rats were imaged twice a week as described above. Addition of Dox to the drinking water was performed at 14 days, 8 months, and 13 months post-aav injection Statistical analysis In vitro studies were repeated three times with 3 9 replicates. A representative experiment is shown in Fig. 2-1 (n = 9). In vivo studies were conducted and data observed using independent animals, with N = 4 8 for Fig. 2-2 due to animals being sacrificed at different time points during the study and N = 4 for Fig All data are represented as median and interquartile range boxplots with percentiles calculated using Tukey Hinge. The significant level used was p < 0.05, unless otherwise noted. Assumptions of parametric data were tested for all data: normality of data distribution using Shapiro-Wilk test and homogeneity of variance using Levene s test. For parametric data (Fig. 2-5), one-way analysis of variance (ANOVA) with post-hoc Tukey was 19

40 2.3.1 In vitro response of rat articular chondrocytes to AAV2 vectors GFP expression was visualized in articular chondrocytes infected with AAV-CMV-GFP as early as 24 h post-infection (data not shown). Articular chondrocytes infected with AAV-CMV-Luc had luciferase expression (Fig. 2-1). Infection of articular chondrocytes with AAV-TRE-Luc led to weak bioluminescence when no Dox was added (Fig. 2-1, AAV-TRE-Luc). This signal was significantly increased when the cells were cultured with Dox at concentrations of 1 μg/ml and 2 μg/ml for 36 h (Fig. 2-1, *p < for AAV-TRE-Luc: 1 μg/ml Dox and AAV-TRE-Luc: 2 μg/ml Dox compared to AAV-TRE-Luc). The bioluminescence observed in vitro with the AAV-TRE-Luc vector and addition of Dox was also greater than that obtained with the AAV- CMV-Luc vector (Fig. 2-1, #p < for AAV-TRE-Luc: 1 μg/ml Dox and AAV-TRE-Luc: 2 μg/ml Dox compared to AAV-CMV-Luc). A significant difference was also observed between the two concentrations of Dox tested (Fig. 2-1, p < for AAV-TRE-Luc: 2 μg/ml Dox compared to AAV-TRE-Luc: 1 μg/ml Dox) Duration of in vivo transgene expression after a single intra-articular injection of AAV2 The IVIS 200 optical imaging system enables in vivo tracking of luciferase expression in the same rat knee joints over a 1 year period. Bioluminescence was visualized as early as 14 days after intra-articular injection of AAV-CMV-Luc and persisted in all animals for 1 year, the study end point. Figure 2-2A shows bioluminescent images from a representative animal at 14 days, 1 month, 6 months, and 1 year after intra-articular injection of AAV-CMV-Luc. In all study animals, the luciferase signal remained constant throughout the duration of the study (Fig. 2-2B). 21

41 The contralateral AAV-CMV-GFP-injected knees did not show luciferase signal during the study. Intraperitoneal injection of luciferin and imaging before sacrifice also indicated that no luciferase signal was present outside the injected knee area. Figure 2-2: Duration of in vivo transgene expression after a single intra-articular injection of AAV-CMV- Luc Transgene expression was observed and quantified using bioluminescence imaging to detect luciferase activity. (A) Bioluminescent images of the left knee joint of a representative rat at 14 days, 1 month, 6 months, and 1 year after a single intra-articular injection of AAV-CMV-Luc. (B) Median and interquartile range of bioluminescence observed in the left knee joint of all rats in the study over 1 year following intra-articular injection of AAV-CMV- Luc. N = 4 8 rats (N = 8 at 14 days, N = 6 at 1 and 2 months, N = 5 at 3 months, and N = 4 from 4 months to 1 year). No significant difference was observed between the different time points Localization of in vivo transgene expression after a single intra-articular injection of AAV2 To localize luciferase signal, knees were imaged intact (Fig. 2-3A) and after exposing the intraarticular space (Fig. 2-3B). Bioluminescence was concentrated in two areas (Fig. 2-3B and C). The majority of the signal was found in the exposed infrapatellar fat pad area, with some signal 22

42 in the intra-articular space containing the ligaments and meniscus. No bioluminescence was observed in articular cartilage. Figure 2-3: Localization of in vivo luciferase expression after a single intra-articular injection of AAV- CMV-Luc by using the IVIS optical imaging system (A) Luciferase was detected in the intact left knee joint. (B) Exposure of the intra-articular space of the same knee joint in (A) revealed that the luciferase signal was located in two major areas. (C) A higher magnification of (B) indicated that the signal was mainly found in the infrapatellar fat pad and in the soft tissues between the femur and tibia, such as the ligaments and meniscus. Tissues retrieved from the AAV-CMV-GFP-injected side provided more information on the localization of cells expressing the transgene. Abundant GFP-positive cells were found in soft tissues such as synovium, infrapatellar fat pad, and tissues within the intra-articular joint 23

43 space. Figure 2-4A shows a large area of GFP-positive cells within meniscus, while Figure 2-4B shows GFP-positive cells in soft tissues surrounding the tibial plateau. GFP-positive cells could be found when compared to soft tissues. As seen in Figure 2-4C, two small areas containing GFP-positive cells were found on the trochlear ridge of the femur. Large areas of GFP-positive cells in articular cartilage were not found in any of the study animals Controlled in vivo transgene expression following a single intra-articular injection of AAV2 Persistent luciferase expression was observed for the duration of the experiment, in all knee joints injected with AAV-CMV-Luc (Fig. 2-5A, B, and C). Intra-articular injection of AAV- TRE-Luc led to a weak luciferase signal before addition of Dox to the drinking water of the rats (Fig. 2-5A). Fourteen days post-aav injection, Dox was added to the drinking water and led to a significant increase in the luciferase signal after 7 days (Fig. 2-5B and D, median fold increase of and interquartile range of (twenty-fifth-percentile = 8.20, seventy-fifth-percentile = 19.46) and *p = compared to Day 0: Dox (-)). Removal of Dox significantly decreased the signal 7 days later (Fig. 2-5C and D, #p = compared to Day 7: Dox (+)). When measured 8 months post-aav injection, addition of Dox to the drinking water of the rats for 7 days increased luciferase expression by a median fold increase of 4.31 and interquartile range of 5.45 (twenty-fifth-percentile = 2.16, seventy-fifth-percentile = 6.04) and p = compared to Day 0: Dox (-). The luciferase signal returned to pre-dox values after the inducer had been removed for 7 days. The induction cycle performed 13 months after the initial injection of AAV also showed increased luciferase signal on the AAV-TRE-Luc side 7 days after addition of Dox to the drinking water (Fig. 2-5E, median fold increase of and interquartile range of

44 Figure 2-4: GFP-positive cells in tissues harvested from the knee joint of rats injected with AAV-CMV- GFP, imaged with a stereomicroscope (A) Brightfield image (left panel) of meniscal tissue with the area located within the square enlarged to show the GFP-positive cells (right panel). (B) Brightfield image (left panel) of tibial plateau. Area within the square is magnified in the right panel and shows GFP-positive cells within the soft tissue. (C) Brightfield image of the trochea (left panel). GFP-positive cells were sparse in articular cartilage. Two small areas with GFP-positive cells were found on the trochlear ridge in this animal (right panel). 25

45 (twenty-fifth-percentile = 27.48, seventy-fifth-percentile = 54.90) and *p = compared to Day 0: Dox (-)). Removal of Dox decreased the signal to baseline levels (Fig. 2-5E, #p = compared to Day 7: Dox (+)). Figure 2-5: Controlled in vivo transgene expression following a single intra-articular injection of AAV2 (A-C) Bioluminescent images of a representative rat that received AAV-TRE-Luc in the right knee and AAV- CMV-Luc in the left knee 14 days prior. Images shown were taken (A) before addition of Dox to the drinking water (Day 0), (B) 7 days after Dox was added to the drinking water (Day 7) and (C) 7 days after Dox was removed from the drinking water (Day 14). Luciferase expression was stable in the knee joint injected with AAV-CMV-Luc, but varied with Dox administration in the knee joints injected with AAV-TRE-Luc. (D) Bioluminescence measured in the knee joint 14 days post-aav-tre-luc injection. Bioluminescence is represented as a median fold increase over Day 0, when no Dox was present in the drinking water, and interquartile range. Luciferase expression increased with addition of Dox to the drinking water after 7 days (Day 7: Dox (+)). *p = compared to Day 0: Dox (-). After removal of Dox from the drinking water for 7 days (Day 14: Dox (-)), the luciferase signal decreased. #p = compared to Day 7: Dox (+). (E) Median and interquartile range of bioluminescence measured 13 months postinjection with AAV-TRE-Luc. Luciferase expression increased 7 days after Dox was present in the drinking water (Day 7: Dox (+)). *p = compared to Day 0: Dox (-). Luciferase expression decreased 7 days after removal of Dox (Day 14: Dox (-)). #p = compared to Day 7: Dox (+). N = 4 rats. 2.4 DISCUSSION Prevention of articular cartilage degradation or promotion of its repair in inflammatory and degenerative arthritis remains challenging. Therapeutic strategies that rely on the delivery of 26

46 growth factors to enhance repair or inhibitory molecules that would prevent degradation are of interest. Ideally, the therapeutic protein should be produced in situ, to obtain a persistent and stable concentration, thus limiting the need for purification processes, repeated injections, and supra-physiological doses, which are not cost effective. Gene therapy is a promising alternative to the delivery of therapeutic factors, as it provides the genetic material necessary for protein synthesis in situ. In this study, we analyzed important aspects for efficient intra-articular gene delivery. These included the persistence and localization of transgene expression after a single intra-articular injection of AAV2, as well as the ability to control the transgene expression over time when using an inducible AAV2 vector. Efficacy of AAV2 for in vitro chondrocyte transduction was evaluated using GFP and luciferase as reporter genes. In accordance with other studies on mice, rabbit, equine, and human chondrocytes (98, ), rat articular chondrocytes were also capable of uptaking the AAV2 vector and producing the transgene protein, suggesting that AAV2 could be used to infect rat chondrocytes in vivo. The ability to induce transgene expression with addition of Dox to the cell culture medium demonstrated the potential to control the timing of transgene expression. Studies in nonhuman primates indicate that administration of AAV to the brain leads to transgene expression for as long as 8 years (100), yet few studies have assessed the persistence of transgene expression after AAV administration to the rat knee joint past a few months (45, 79, 95, 99). Intra-articular injections into the rat stifle joint, while challenging, have previously been reported by our group and others (45, 46, 79, 105). Given this experience, we believe that AAV was delivered intra-articularly in this study, although it is possible that some leakage or partial extra-articular injection may have occurred. By using bioluminescence, the same animal could be imaged repeatedly, providing a clear profile of transgene persistence and 27

47 biodistribution. All animals had stable luciferase transgene expression for up to 1 year following intra-articular injection of AAV-CMV-Luc. A recent study employing recombinant lentivirus or adenovirus vectors did not observe such stable transgene expression in knees of immunocompetent rats (106). They observed strong transgene expression that diminished over a 3-week period. Injection of the same vectors into athymic rats led to an initially high transgene expression, which decreased to 20% and remained at that level for 6 months (106). Prolonged transgene expression in the current study suggests lower immunogenicity of AAV2 than lentivirus or adenovirus. Stability of transgene expression following AAV2 injection into the rat knee joint supports a potential role for AAV2 gene therapy in treatment strategies for inflammatory or degenerative arthritis by facilitating the synthesis of therapeutic factors within the joint space. Consistent with previous reports of AAV injection into the knee joint of various animals, both luciferase and GFP transgene expressions were mostly localized to soft tissues (44, 46, 95, 97, 99). GFP imaging with the stereomicroscope provided a more precise localization than the bioluminescence imaging system and indicated that few sparse areas of GFP-positive cells could be found in articular cartilage. A recent study employing selfcomplementary AAV2 injected into the intra-articular joint of guinea pigs also demonstrated transgene expression by articular chondrocytes (97). Previous studies using AAV have shown higher levels of transgene expression in diseased cartilage compared to normal cartilage (44, 46). Seeing that intact and undamaged articular cartilage is unlikely to require treatment, future studies will test AAV in rat models of articular cartilage damage, as it will more closely mimic the osteoarthritic environment. Abundant transgene expression in soft tissues suggests that cells within these tissues, such as those within the infrapatellar fat pad, can uptake the AAV2 and lead 28

48 to transgene expression. Given the low turnover rate of these cells, intra-articular delivery of AAV2 targeted to soft tissues is of value for anti-inflammatory treatment strategies for arthritis conditions. For articular cartilage repair, it may be necessary to target more specifically the chondrocytes. This may be especially important if delivering genes encoding for bone morphogenetic proteins, as their expression in soft tissues could result in ectopic mineralization. We and other are exploring strategies for targeted delivery of transgenes to articular chondrocytes and chondral defects (42, 107, 108). Lack of luciferase signal in the contralateral control knee that did not receive AAV- CMV-Luc, and lack of signal anywhere else in the animal suggests that intra-articular injection limits AAV2 delivery to tissues within the joint and does not travel systemically. This is advantageous, as bioactive factors present from viral transgene expression would not affect tissues outside of the joint, and possibly lead to negative side effects. The dose of AAV used in this study of 2.5 x vg/knee joint was chosen based on what was previously been shown to be effective in rats (45). This prior study employed vg of AAV/rat knee joint and reported transgene expression for at least 100 days. The dose chosen for our current rat study will need to be increased in order to be effective in a human joint, which is larger in size and in volume of synovial fluid, resulting in a larger volume of distribution. Others have shown that it is feasible to administer AAV to humans through intra-articular injection. Human clinical trials for rheumatoid arthritis using a recombinant, single-stranded AAV2 vector have performed intra-articular injections at doses ranging from to vg/ml and 5 ml was injected per joint (88, 109). The authors reported these dosages to be safe and feasible, and although some improvement in patient-reported outcomes was observed, the transgene expression was not found to be sufficient. It should be noted that humans have serum 29

49 IgG and neutralizing factors against different types of AAV, representing a limiting factor to AAV gene therapy (110). In moving towards direct intra-articular injection of AAV to larger joints, it will be important to determine the optimal dose of AAV to deliver and the serotype that will face the least amount of neutralizing antibodies. Studies involving inducible gene therapy for musculoskeletal application have mainly focused on genetic engineering of cells ex vivo, followed by their implantation in vivo (75, ). Direct viral gene delivery is easier to administer, and does not require the additional time and cost associated with ex vivo cell-mediated gene delivery. As shown in this study, the transgene expression can last for up to a year, which is not the case with implanted cells (42). By persisting for a prolonged period of time, there is the possibility to perform multiple cycles of transgene expression with a single injection of AAV, as shown in this study. We observed increased transgene expression with addition of Dox to the drinking water and reduction of transgene expression following its removal at both 14 days and 13 months post- AAV injection. A similar trend was observed after 8 months. These data demonstrate the persistence of the transgene and controllability of its expression. The variability in observed transgene expression between the 14 days, 8 months, and 13 months time points could reflect type II error due to the small sample size or potentially be related to a difference in Dox intake. Adding Dox to the drinking water, while simple and shown in this study to be capable of controlling in vivo intra-articular transgene expression, may however lead to some degree of variability since free water intake is less well controlled than oral administration as a pill or measured suspension. Future studies with inducible gene expression systems may need to use direct injection of Dox in small animals and oral suspension or pill administration in larger animals. 30

50 Induction of gene expression 13 months post-aav injection is of clinical relevance when a therapeutic protein needs to be re-administered to further promote cartilage repair, or in cases where cartilage resumes degrading or inflammation increases and another round of treatment is necessary. An inducible AAV2 gene expression system is also of interest for the delivery of multiple factors. Different biological molecules could be synthesized at different time points, depending on the regulatory transcriptional activator. This would provide a controlled environment for cartilage repair, where multiple growth factors are involved. A limitation of the inducible AAV2 gene expression system used in this study is the observed leakiness without the addition of Dox. This can potentially be addressed in future studies by newer rtta vectors that have been developed to reduce basal activity and that require less Dox to be activated (114). 2.5 CONCLUSIONS In summary, AAV2 delivered by a single intra-articular injection to a normal rat knee joint led to persistent and stable transgene expression that was mainly localized to soft tissues of the joint, with some expression in chondrocytes. Intra-articular injection of an inducible AAV2 vector demonstrated the ability to regulate in vivo transgene expression by oral administration of Dox, a compound that is safely used in humans. This regulation was possible immediately after delivering the AAV to the knee joint, and 13 months later. Ability to achieve stable yet high regulatable long-term intra-articular transgene expression is of potential clinical utility for development of new treatment strategies for intra-articular disease processes, such as inflammatory and degenerative arthritis. 31

51 3.0 PERSISTENCE, LOCALIZATION, AND EXTERNAL CONTROL OF TRANSGENE EXPRESSION AFTER SINGLE INJECTION OF ADENO-ASSOCIATED VIRUS INTO INJURED JOINTS 3.1 INTRODUCTION Traumatic and degenerative injuries to articular cartilage are a leading source of disability. The limited healing potential of articular cartilage often leads to development of osteoarthritis (OA). OA is a chronic and progressive disease, and patients need to be on medications for a long period of time. Common medications for OA include non-selective non-steroidal anti-inflammatory drugs (NSAIDs) (115, 116). Chronic and often daily use of these drugs; however, is not without significant complications. NSAID therapy results in systemic side effects and is responsible for 16,500 deaths per year in the US (117). As well, there are no disease modifying treatments for osteoarthritis nor a method to completely re-establish cartilage function (79). Cartilage injuries or OA affects only a limited number of joints, without major extra-articular or systemic symptoms. Hence, delivery of bioactive agents in a localized manner to the injured joints is an attractive therapeutic option. It is therefore of critical interest to investigate an effective delivery vehicle that targets articular joints. A potentially novel and innovative treatment strategy is gene therapy, utilizing viral vectors, such as adeno-associated virus (AAV) to deliver therapeutic proteins of interest to 32

52 affected joints to enhance joint repair or to prevent cartilage degradation. Recombinant AAV is a promising delivery vehicle for articular cartilage with the advantages of sustained transgene expression, reduced potential for host immune response, and the capacity to transduce both dividing and non-dividing cells in vivo and in vitro (108, 118). The viral vector is derived from an endemic, non pathogenic parvovirus, and intra-articular injection of AAV has been used in clinical trials for delivery of bioactive substances (88, 119). Numerous serotypes of AAV exist, each serotype with different preferential targets (44). AAV5 has recently been suggested to have better transduction efficiency in rodent arthritic joints; however, AAV2 has the best defined safety profile as it is used in clinical trials (48). A recently published study from our laboratory has shown both stable and persistent reporter transgene expression at one year after a single intra-articular injection of AAV (47). Most of the AAV transduction occurred in the intra-articular soft tissues covered with synovial lining, with limited articular chondrocyte transduction. This is consistent with other studies, as vector penetration through the dense cartilage extracellular matrix is restricted (44, 116). However, high level expression of transgene product was possible from the soft tissues. Nonetheless, there are potential safety issues concerning long-term or high-dose expression of transgenes, in that overexposure could result in undesirable side effects. For this reason, controllable vectors have been developed to limit treatment time/dosage to the minimal needed to achieve therapeutic efficacy. One novel system is the Tetracycline (Tet)-on promoter, in which control is facilitated by using tetracycline molecule as an activator of the promoter, stimulating transgene expression only in the presence of the drug (76). Previous studies in our laboratory have successfully modulated gene expression utilizing the Tet-on system and doxycycline, a tetracycline analog (47). However, as intact/undamaged articular cartilage is 33

54 (pen/strep, Gibco) and incubated for 2 3 days in 37 C, 5% CO 2 in 1:1 ratio Dulbecco s modified Eagle medium (DMEM) and F-12 (DMEM/F-12, Gibco), 10% fetal bovine serum (FBS, Gibco), 2% pen/strep, and 1% amphotericin B (Invitrogen). The explants were divided into three groups in triplicate: No Injury/No AAV Control, No Injury/AAV-CMV-Luc, Scratch injury with AAV-CMV-Luc. The explants from the latter group had the femoral condyles scratched with a blade. The explants from the No Injury/AAV-CMV-Luc and Scratch/AAV- CMV-Luc groups were transduced with AAV-CMV-Luc at a concentration of 80,000 vg/cell in serum-free DMEM/F-12. After one hour incubation, the culture media was replaced with fresh DMEM/F-12, 10% FBS, 2% pen/strep, and 1% amphotericin B. The culture media was changed after two days. The explants were imaged after 96 hours post-aav transduction, using IVIS Imaging System 200 (Xenogen) by adding 10 μl of 30 mg/ml D-Luciferin Potassium Salt (Caliper Life Sciences) per 1 ml of culture media. The images were taken at 1 min exposure Animal studies Animal experiments were performed following a University of Pittsburgh Institutional Animal Care and Use Committee (IACUC)-approved protocols and Longitudinal in vivo study was performed using forty-eight three month old male Sprague Dawley rats Animal surgeries Twenty-four rats underwent unilateral OCD surgeries, while the other twenty-four rats underwent ACLT surgeries as previously described (121, 122). Briefly, all the procedures were carried out while the animals were anesthesized with inhalation of isoflurane (Phoenix 35

55 Pharmaceuticals): 2-5% for induction and % for maintenance. Rat knee joints were exposed via medial parapatellar and medial patellar tendon approaches. The patella was dislocated laterally and the knee was flexed. For the OCD surgeries, the midpoint of the femoral trochlea was identified and a 1.5 mm carbide tipped drill (Emil Lange/ELA) was used to create an OCD approximately 1.0 mm deep. The subchondral bone was penetrated and bleeding bone was observed. For the ACLT surgeries, ACL and posterior cruciate ligament (PCL) were visualized with the aid of a 3x surgical loupe (Carl Zeiss). Forceps were used to protect the PCL and the ACL was severed using a 1.5 mm Cutting Edge Beaver Needle Blade (Becton, Dickinson and Company). ACLT was confirmed with an anterior drawer test. Following injury creation for both OCD and ACLT, the joint spaces were washed with sterile saline, and both capsule and skin were sutured using monofilament 4-0 monocryl (Ethicon). An intramuscular injection of mg/kg buprenorphine (Hospira) was administered twice per day for two days following surgery for analgesia AAV injections AAV2-CMV-Luc was used to characterize the persistence and localization of the transgene signal while AAV2-TRE-Luc was used to characterize the external controllability of the transgene signal using oral doxycycline. Two different AAV injection time points were investigated to compare the transgene expression levels: post-injury AAV injection and pre-injury AAV injection. For the post-injury AAV injection group, the viral vectors were injected bilaterally into both injured and intact joints at one week post-injury for OCD rats and three weeks post-injury for ACLT rats. For the preinjury AAV injection group, the AAVs were injected bilaterally into the joint. After twenty-eight 36

56 days, when the transgene expression level has stabilized, the animals underwent unilateral joint surgeries, either OCD or ACLT. Each knee was given a total of 50 μl of AAVs, containing 2.5 x vg of either AAV2- CMV-Luc or AAV2-TRE-Luc and an equal amount of AAV2-CMV-rtTA by single intraarticular injection. During the injection procedures, animals were anesthesized with gaseous isoflurane. Six rats were used for each viral construct per injury model per AAV injection time points (Table 3-1). Table 3-1: Animal study design Two AAV-injection time points were investigated on OCD and ACLT models of joint damage, with N = 6/injury type/aav-injection time point/aav-construct. AAV-injection Timepoint Injury AAV2 construct N Post-injury OCD AAV2-CMV-Luc 6 AAV2-TRE-Luc 6 ACLT AAV2-CMV-Luc 6 AAV2-TRE-Luc 6 Pre-injury OCD AAV2-CMV-Luc 6 AAV2-TRE-Luc 6 ACLT AAV2-CMV-Luc 6 AAV2-TRE-Luc 6 Total number of rats: External control of transgene expression To induce expression of luciferase in the animals injected with AAV2-TRE-Luc, the rats were administered drinking water containing doxycycline (Sigma) at a concentration of 2 mg/ml for seven days, followed by its removal. To ensure fresh supply of doxycycline, the water was replaced every day and protected from light using amber drinking bottles. Addition of doxycycline to the drinking water was performed at 14 days, 3 months, and 6 months post-aav injection. Rats injected with AAV2-CMV-Luc had regular drinking water for the duration of the study. 37

57 3.2.7 Longitudinal in vivo live animal imaging At each imaging time point, rats were anesthetized with isoflurane inhalation, followed by a 50 μl intra-articular injection of 30 mg/ml D-Luciferin Potassium Salt substrate. After 5 minutes, rats were placed in and imaged with an IVIS 200 optical imaging system. Rats injected with AAV2-CMV-Luc were imaged every week for the first month, and monthly thereafter, until six months post-aav injection, when the rats were sacrificed. On the day of sacrifice following regular imaging as detailed above, rats received a 400 μl intra-peritoneal injection of 30 mg/ml D-Luciferin Potassium Salt substrate (Goldbio) to determine if any signal was present outside the joint. The joint was then opened to image the intra-articular tissues. Rats injected with AAV2- TRE-Luc were imaged three times for every oral doxycycline stimulation cycle: pre-doxycycline time point prior to addition of doxycycline, 7 days following doxycycline addition, and 7 days following doxycycline removal. These rats were sacrificed at six months post-aav injection as well, following the third doxycycline stimulation cycle. All the rats were sacrificed via CO 2 asphyxiation and subsequent thoracotomy Statistical analyses All the statistical analyses were performed using Statistical Packages for Social Studies (SPSS) 17.0 (IBM). Results were judged for significance at p <.05, unless otherwise noted. Explant data were observed in triplicate (Fig. 1, n = 3). In vivo animal data were observed using independent animals, with N = 6 per group. All the data were tested for assumptions of parametric data: Shapiro-Wilk for normality of data distribution and Levine s for homogeneity of variance. The parametric bioluminescence data for the explant study is represented as median and interquartile 38

58 range boxplots, and analyzed using one-way independent analysis of variance (ANOVA) with post hoc Tukey (Fig. 3-1). The longitudinal bioluminescence data for the animal studies assessing the persistence and magnitude of the AAV-transgene are represented as line graphs ± standard error of mean (SEM) and were analyzed using two-way mixed ANOVA, with main independent variables of joint injury status as paired variable and time as repeated variable (Fig. 3-2 and 3-4). Friedman s ANOVA test with a Bonferroni correction was used as a post hoc test to assess effect of time on each of the injured and intact joints (p <.025 considered significant) and Wilcoxon Signed Ranks test with a Bonferroni correction was used as a post hoc test to assess the effect of joint injury status at each outcome time points when the interaction effect of time between the time and joint injury status was significant (Fig. 3-4B). The bioluminescence data assessing controllability of transgene expression were analyzed using one-way repeated measured ANOVA (Fig. 3-6). 3.3 RESULTS In vitro AAV transduction of explants Transgene expression was observed from femoral cartilage explants transduced with AAV- CMV-Luc (Fig. 3-1). The bioluminescence signal was greater in No Injury/AAV-CMV-Luc group (p =.046) and Scratch/AAV-CMV-Luc group (p =.008) compared to No Injury/No AAV Control group. While no difference was observed between the two AAV-CMV-Luc transduced groups (p =.313), the mechanical scratch injury model where the condyles were scratched with a blade showed brighter bioluminescence at both lateral and medial condyles. 39

59 Figure 3-1: Explant AAV transduction of rat femoral cartilage Rat femoral cartilage explants were either scratched or kept intact (No Injury), after which they were transduced with AAV-CMV-Luc. Luciferase signal was detected after 96 h. (A) Representative images of bioluminescence signal from the explants. (B) Median and interquartile range of bioluminescence for the different explant groups. n = 3. *p <.05 versus No Injury/No AAV Control group AAV transduction in osteochondral defect injury AAV transgene expression was persistent in both the OCD and intact joints whether the AAV was injected post- or pre-ocd injury (Fig. 3-2). However, the magnitude of the transgene expression levels in the OCD joints was different between the two AAV injection time points. For the knees with AAV injected post-ocd, there was a significant main effect of the injury status on the bioluminescence level (p =.015), where the OCD knees had lower signal than the intact knees (Fig. 3-2B). On the other hand, for the knees with AAV injected pre-ocd, the main effect of the injury status on the bioluminescence level was non-significant (p =.924, Fig. 3-2D). As well, the luciferase expression remained unchanged from the pre-ocd time points. For both 40

60 the AAV injection time point groups, there was a non-significant main effect of time on the bioluminescence level (p =.175 for post-ocd AAV injection and p =.185 for pre-ocd AAV injection): the transgene magnitude remained constant throughout the duration of the study for each of the joints. The interaction effect between the joint injury status and the time was nonsignificant in both cases as well (p =.182 or post-ocd AAV injection and p =.921 for pre-ocd AAV injection). Figure 3-2: Persistence of AAV transgene in osteochondral defect injury model Persistence of in vivo transgene expressions in OCD injured joints with a single intra-articular injection of AAV- CMV-Luc post-ocd (A and B) or pre-ocd (C and D) were assessed via bioluminescence. (A) Representative images of transgene signal from the OCD and intact joints at 7 days, 1 month, and 6 months following AAV-CMV- Luc injection 1 week after OCD injury. (B) Mean and SEM of bioluminescence observed in the OCD and intact joints of all rats in the group over 6 months following AAV-CMV-Luc injection 1 week after OCD injury. (C) Representative images of transgene signal from the OCD and intact joints injected with AAV-CMV-Luc 4 weeks prior to OCD injury at pre-ocd, 1 month, and 6 months time points. (D) Mean and SEM of bioluminescence observed in the OCD and intact joints of all rats in the group over 6 months following OCD injury in joints injected with AAV-CMV-Luc 4 weeks prior. N = 6/group. Upon exposing the intra-articular space, the location of transgene expression was different in the OCD injured knees between the two different AAV injection time points (Fig. 3-3). For both of the AAV injection time points, the intact joints had the diffuse and high magnitude signal in the exposed infrapatellar fat pad. However, for the OCD injured joints, the 41

61 bioluminescence signal was concentrated to the injured cartilage and the associated OCD repair tissue in the post-ocd AAV-injected knees (Fig. 3-3A), whereas the signal was more diffusely found in intra-articular soft tissues, mainly the fat pad in the pre-ocd AAV-injected knees, with no bioluminescence observed in the injured cartilage (Fig. 3-3B). Hence, the pre-ocd AAVinjected knees had transgene signal location similar to those of the intact joints. Figure 3-3: Localization of AAV transgene in osteochondral defect injury model Intra-articular localization of transgene expressions in OCD injured joints with a single intra-articular injection of AAV-CMV-Luc were assessed after exposing the joint on the day of sacrifice using the IVIS optical imaging system. (A) Representative intra-articular images of transgene signal from the exposed OCD and intact joints that were injected with AAV-CMV-Luc 1 week after OCD injury. (B) Representative intra-articular images of transgene signal from the exposed OCD and intact joints that were injected with AAV-CMV-Luc 4 weeks prior to OCD injury AAV transduction in anterior cruciate ligament transection injury Similarly to the OCD injury model, the transgene expression was persistent in both the ACLT and intact joints, and for both post-aclt and pre-aclt AAV injections (Fig. 3-4). The magnitude of the bioluminescence levels in the ACLT joints was again different between the two AAV injection time points. For the joints with AAV injected post-aclt, there was a significant main effect of the injury status (p =.001): the ACLT joints had lower signal than the intact (Fig. 3-4B). For the knees with AAV injected pre-aclt, on the other hand, the main effect of the injury status was non-significant, with no change in transgene expression from the pre-aclt time points (p =.717, Fig. 3-4D). The main effect of time on the bioluminescence level was 42

62 significant for the post-aclt AAV injected group (p =.010) and non-significant for the pre- ACLT AAV injected group (p =.401). The interaction effect between the joint injury status and the time was significant for post-aclt AAV injection study group (p =.011) and nonsignificant for pre-aclt AAV injection study group (p =.872). Further post hoc testing of the post-aclt AAV injection study group showed that the effect of time on the bioluminescence level of the ACLT joints is non-significant for the ACLT injured joints (p =.992). Hence, the transgene magnitude remained constant throughout the study period in the ACL injured joints for both post-aclt and pre-aclt AAV injection groups. Figure 3-4: Persistence of AAV transgene in anterior cruciate ligament transection injury model Persistence of in vivo transgene expressions in ACL transected joints with a single intra-articular injection of AAV- CMV-Luc post-aclt (A and B) or pre-aclt (C and D) were assessed via bioluminescence. (A) Representative images of transgene signal from the ACLT and intact joints at 7 days, 1 month, and 6 months following AAV-CMV- Luc injection 3 weeks after ACL transection. (B) Mean and SEM of bioluminescence observed in the ACLT and intact joints of all rats in the group over 6 months following AAV-CMV-Luc injection 3 weeks after ACL transection. (C) Representative images of transgene signal from the ACLT and intact joints injected with AAV- CMV-Luc 4 weeks prior to ACL transection at pre-aclt, 1 month, and 6 months time points. (D) Mean and SEM of bioluminescence observed in the ACLT and intact joints of all rats in the group over 6 months following ACL transection in joints injected with AAV-CMV-Luc 4 weeks prior. N = 6/group. When the intra-articular space was exposed, the localization of bioluminescence signal was different in the ACLT joints between the two different AAV injection time points as well 43

63 (Fig. 3-5). Consistent with the intact sides of the OCD injury model animals, the intact joints showed diffuse signal in the exposed infrapatellar fat pad for both of the AAV injection time points. As well, the diffuse bioluminescence signal was found in the infrapatellar fat pad for the pre-aclt AAV-injected ACL transected joints, with no signal found in the cartilage (Fig. 3-5B). However, for the post-aclt AAV-injected ACL transected joints, the bioluminescence signal was lower in the intra-articular soft tissues but some region of concentrated signal in the injured cartilage (Fig. 3-5A). Figure 3-5: Localization of AAV transgene in anterior cruciate ligament transection injury model Intra-articular localization of transgene expressions in ACL transected joints with a single intra-articular injection of AAV-CMV-Luc were assessed after exposing the joint on the day of sacrifice using the IVIS optical imaging system. (A) Representative intra-articular images of transgene signal from the exposed ACLT and intact joints that were injected with AAV-CMV-Luc 3 week after ACL transection. (B) Representative intra-articular images of transgene signal from the exposed ACLT and intact joints that were injected with AAV-CMV-Luc 4 weeks prior to ACL transection External control of AAV-transgene expression in injured joints Rats that received the inducible AAV2-TRE-Luc showed gene expression upregulation with addition of Dox and downregulation following its removal from the drinking water from both the OCD and ACLT injured joints (Fig. 3-6). This finding was consistent between the two AAV injection time points. 44

64 Figure 3-6: Controllability of AAV transgene in joint injury models Intra-articular transgene expressions in injured joints with a single intra-articular injection of AAV-TRE-Luc postinjury (A and C) or pre-injury (B and D) were assessed after a cycle of doxycycline addition and removal using the IVIS optical imaging system. (A) Representative intra-articular images of transgene signal from the OCD joints that were injected with AAV-TRE-Luc 1 week after OCD injury. (B) Representative intra-articular images of transgene signal from the OCD joints that were injected with AAV-TRE-Luc 4 weeks prior to OCD injury. (C) Representative intra-articular images of transgene signal from the ACLT joints that were injected with AAV-TRE-Luc 3 weeks after OCD injury. (D) Representative intra-articular images of transgene signal from the ACLT joints that were injected with AAV-TRE-Luc 4 weeks prior to ACL transection. *p < 0.05 compared to Day 0: Pre-Dox. #p < 0.05 compared to Day 7: Dox +. N = 6 rats. 3.4 DISCUSSION Treatment or prevention strategies of pathologic processes within articular joints are severely limited. Hence, a successful delivery strategy for adequate levels of therapeutic molecules in a localized manner is critically important. Gene therapy strategies, therefore, has gained a lot of attention for arthritis treatment (44, 116). Numerous vectors are available for in vivo gene transfer, such as naked DNA, retrovirus, adenovirus, etc.; however, most are limited in term of 45

65 safety, efficacy, and duration (123). AAV, on the other hand, is a very promising vector system that may provide the best balance between safety and in vivo gene delivery efficiency (116). Methods to localize and control in vivo transgene expression are important to enhance the therapeutic efficiency and safety of intra-articular AAV gene therapy. This has been achieved in intact joints (47). However, to more closely mimic the osteoarthritic environment, AAV transduction to injured cartilage and joints has been investigated. We initially characterized the efficacy of AAV transduction in explants culture of femoral cartilage. With 80,000 vg/cell AAV2-CMV-Luc, significant transduction was observed for rat femoral explants. There was no significant difference with transduction efficacy between the two AAV-transduced groups; however, the mechanical injury model where the condyles were scratched with a blade showed high intensity of luciferin flux at both lateral and medial condyles. This is potentially due to the scratch injury creating a more permeable path to articular chondrocytes for AAV transduction. Thus, we expected to have more AAV-transduction in damaged areas of articular cartilage. For the in vivo animal studies, we chose two commonly used joint injury models of high clinical relevance: OCD and ACLT. The timing of AAV injection, pre- or post-joint injury, permitted us to characterize the effect of injury on AAV transduction and localization of transgene expression. For both of the injury models, AAV transduces and is mainly expressed by intra-articular soft tissues in the intact sides, as shown in the previous study (47). However, if the AAV is injected after joint injury, as would likely be the case in a clinical setting, the transgene signal localizes to the vicinity of the injured cartilage and associated repair tissue. As in the case for the explants study, the injured cartilage, with more permeable extracellular matrix, would have allowed AAV to travel and traduce the chondrocytes/injury activated repair cells. The 46

66 transgene expression from the soft tissues was limited for both OCD and ACLT injured joints. This can also be due to a number of reasons, such as increased volume of synovial fluid in injured joints diluting the intra-articular viral concentrations, injury-activated macrophages clearing up the virus prior to effective transduction, or the viral gene promoter shut off in response to the injury (124). When the AAV is injected before injury, the transgene signal magnitude as well as localization was no different from those of the intact joints. All of the transgene expression was found in the soft tissues with no detectable signal from the injured cartilage or associated repair tissue. This pre-injury AAV injection data makes the theory that promoter shuts off in response to injury less likely. The differential transgene location between the two AAV-injection timepoints can be used to optimize AAV-mediated cartilage repair strategies. The highly localized gene expression following AAV injection post-injury is advantageous for gene therapy strategies that deliver anabolic growth factors that would aid in cartilage repair, especially genes that need to specifically target chondrocytes to be effective. Such strategies may include RNA interference against various cartilage catabolites, such as aggrecanases/adamtss (70), or chondrogenic growth factors that have adverse effects on other intra-articular soft tissues, such as transforming growth factor (TGF)-β 1. TGF-β 1 has been shown to increase chondrocyte matrix synthesis when its gene vector was injected in the joint; however, its overexpression from the synovium resulted in adverse fibrosis (66). Diffuse signal, seen when AAV is injected before creation of injury, is also of potential value, as it provides a model to study the delivery of protective factors to individuals that may be at high risk for cartilage degeneration. Although the virus does not specifically transduce chondrocytes, a high level of transgene expression from surrounding soft tissues still allows for 47

67 preventative therapeutic approaches by using the soft tissue as a drug depot to release protective molecules. The high intra-articular concentrations of secreted products, such as a cytokine antagonist like interleukin-1 receptor antagonist protein, can protect the cartilage by competing with catabolic factors in the joint (74). Moreover, since synoviocytes release numerous proinflammatory molecules in arthritic conditions, a strong tropism of AAV for the synovium can be utilized to suppress expression of any of these injurious compounds (125). The controllability of gene expression in intact and injured joints is also important since gene expression may not be needed for a prolonged period of time to have a beneficial effect, and it provides added means of safety. We have shown this controllability in all the injury models, whether the AAV was injected post- or pre-injury using oral administration of doxycycline in drinking water. Doxycycline is an analog of tetracycline, an anti-microbial agent that is safely used in humans (78). In addition, the use of doxycycline as the transgene inducer molecule may have added beneficial effect for cartilage protection. Doxycycline inhibits collagen degrading enzymes, such as matrix metalloproteinases (MMPs), and has shown beneficial effects in both OCD and ACLT injury models by preventing MMP upregulation (121, 126). The studies investigating AAV transduction in injured joints are limited, and only rheumatoid arthritis injury setting has been explored (44, 46, 127). Two groups used a transgenic mice model that spontaneously develops arthritis due to tumor necrosis factor (TNF)-α overexpression (44, 127). Goater et. al. reported increased AAV transduction efficiency with increased degree of the joint disease following an intra-articular injection of AAV, with synoviocytes as the primary target. As well, in vitro transduction efficiencies of synoviocytes extracted from the transgenic mice increased with the treatment of these cells with damaging 48

68 agents, such as irradiation and TNF-α. However, their study was done using single stranded AAV, where the second strand synthesis is the limiting step in AAV transgene expression. Hence, the injurious agents promote DNA synthesis and thus transgene expression as well. The use of double stranded or self complementary AAV has been shown to improve AAV transgene expression (73). Zhou et al. showed persistent transgene expression for 120 days following AAV-Luc injection in the transgenic mice joints; however, they did not compare their bioluminescence level to intact joints, preventing a comparison to our study. Another study investigated intra-articular AAV transduction in Sprague Dawley rats damaged via a joint injection of lipopolysaccharide (LPS) (46). This study showed positive correlations between disease severity and AAV transduction and between disease severity and transgene expression. Again, however, these injury models mimic inflammatory/rheumatoid arthritis rather than osteoarthritis. Limitations of the current investigation include that the models of joint injury were surgically induced, which is removed from most human idiopathic osteoarthritis cases. Hence, the relevance of the results from this study may be more applicable to joint injuries secondary to trauma. However, post-traumatic arthritis, with a known date of onset/injury, is more amenable for early intervention strategies such as AAV-mediated gene therapy compared to idiopathic OA with less definable time of onset (128). Our current focus was mainly for the characterization of AAV-transgene expression in the injured joints. The mechanism behind differential gene expression localization between the post-injury and pre-injury AAV injection time points remains unresolved and needs further elucidation with sham operated animals, as well as various other control groups. 49

69 3.5 CONCLUSIONS While AAV transgene expression was localized to the soft tissues of the uninjured joint and injured joints when AAV was injected pre-injury, AAV was able to transduce articular chondrocytes within the cartilage matrix when injected following injuries. The explant data showing AAV-transduction of articular chondrocytes in damaged cartilage suggests that cartilage matrix injury may have facilitated entry of more AAV into the cartilage. The in vivo data supports use of AAV for persistent, regulated, and safe intra-articular release of bioactive factors from injured joint tissues for cartilage repair/protection. Differential transgene expression localizations can be used to optimize AAV-mediated cartilage repair strategies: post-injury AAV injection approaches would directly target articular chondrocytes in diseased and injured cartilage and pre-injury AAV injection would allow intra-articular delivery of secreted antiinflammatory factors for sustained release from soft tissues to protect the joint. This information can aid in the design of transgene product that could be used for joint strategies. Overall, these data support further study of the in vivo therapeutic potential of AAV for safe and localized delivery of bioactive substances to injured joints. This can aid in cartilage repair, which may delay or prevent the onset of debilitating osteoarthritis. 50

70 4.0 EFFECTS OF DOXYCYCLINE ON MESENCHYMAL STEM CELL CHONDROGENESIS AND CARTILAGE REPAIR 4.1 INTRODUCTION Annually, nearly one million people in the United States are affected by articular cartilage injuries. These injuries result in significant disability and have a substantial disease and financial burden for the healthcare system and the economy. Articular cartilage has limited capability to regenerate or repair following injury, and therefore presents a great therapeutic challenge (129, 130). Furthermore, focal cartilage injuries are known to accelerate the degeneration of surrounding cartilage, hastening onset of osteoarthritis. Microfracture (bone marrow stimulation) is the simplest and most commonly used surgical procedure for many focal cartilage injuries. This technique involves the recruitment of bone marrow derived mesenchymal stem cells (MSCs) to the defect area in an attempt to stimulate chondrogenesis. However, the repair does not restore pristine hyaline cartilage containing type II collagen, but instead results in formation of a mechanically inferior fibrocartilage scar primarily composed of type I collagen (129, 130). Therefore, new and innovative clinical strategies are needed to improve the type and quality of repair tissue in order to prevent the development of osteoarthritis. Doxycycline is a widely available and inexpensive anti-microbial agent that is safely used in humans (78). In addition, doxycycline is a commonly used inducer molecule for the 51

71 tetracycline-inducible gene regulation system for controlled gene therapy applications in the articular joint as well as other organ systems across multiple animal models (47, 131). Besides these anti-microbial and gene-inducing functions, doxycycline may also have intrinsic benefits to cartilage. It has been well established that the tetracycline class of medications have the ability to inhibit matrix metalloproteinases (MMPs), specifically MMP-1 (collagenase-1), MMP-3 (stromelysin), and MMP-13 (collagenase-3), independent of their anti-microbial activity ( ). Doxycycline is the tetracycline analog that is most studied for MMP-inhibition, and it has been shown to exert a beneficial effect against extracellular matrix (ECM) degradation in arthritic diseases, as MMPs are shown to be upregulated in arthritis and following cartilage injury (133, ). Down-regulation of MMPs increases the amount of collagens produced and retained in the ECM (136). However, despite the existing studies suggesting high potential for doxycycline to exert a protective effect against cartilage degeneration, there are limited studies evaluating the effects of doxycycline on the repair tissue formed by MSCs following bone marrow stimulation. This study tests the hypothesis that doxycycline reduces MMP expression, enhances human mesenchymal stem cell (hmsc) chondrogenesis and improves in vivo cartilage repair. 52

73 equipped with a DP71 camera (Olympus). The pellet area was measured using DP2-BSW software (Olympus). The pellets were subsequently fixed in 10% buffered formalin and embedded in paraffin for histological analyses. Five-μm cross-sections were stained with Safranin O-Fast green for sulfated glycosaminoglycan (GAG) and alizarin red for calcium, and immunostained for MMP-13 according to standard protocols. Briefly for MMP-13 immunohistochemistry (IHC), sections were de-paraffined and rehydrated via conventional methods. Antigen retrieval was performed by heating and cooling the slides in 10-mM sodium citrate (ph 6.0) with 0.05% Tween-20 buffer (Sigma-Aldrich) for 20 minutes each at 85 C and room temperature. The sections were then digested in 1 mg/ml hyaluronidase in 0.1 M sodium acetate buffer (both from Sigma-Aldrich) for 30 minutes in 37 C. The hyaluronidase-digested sections were blocked with 10% goat serum (Vector Laboratories) and 1% bovine serum albumin (BSA, Fisher Scientific) for 1 hour. The sections were incubated with a rabbit polyclonal antibody against MMP-13 (Abcam) at 1:100 dilution in 1% BSA solution overnight at 4 C. Endogenous peroxidase was blocked using 0.3% hydrogen peroxide (Sigma) in trisbuffered saline (TBS, Calbiochem) for 15 minutes at room temperature. The slides were then treated with secondary goat polyclonal anti-rabbit antibody (Abcam) in 1% BSA solution in 1:100 dilution for an hour, then developed using ImmPACT AEC Peroxidase Substrate (Vector Laboratories), according to the manufacturer s instructions. The slides were counterstained with hematoxylin (Vector Laboratories). Images of all the stained sections were captured using TE-2000U Eclipse microscope (Nikon) equipped with a DP71 camera. The MMP-13 IHC images in Fig. 4 had the contrast increased by the same value in Adobe Photoshop CS4 to improve MMP-13-positive and -negative cell contrast for print. 54

74 Biochemical analyses After 3, 7, 14, and 21 days of chondrogenic culture, three hmsc pellets per group were washed with phosphate buffered saline (PBS, Gibco) and each dry pellet was frozen at -80 C until biochemical analyses. Each frozen pellet was incubated overnight in 0.25 ml papain buffer (50 mm sodium phosphate buffer (Sigma-Aldrich), 5 mm ethylenediaminetetraacetic acid (EDTA, Gibco), 5 mm cysteine HCl (Sigma-Aldrich), and 0.5 mg/ml papain (Sigma-Aldrich)) in 60 C shaking water bath (200 RPM). The papain-digested pellets were vortexed. GAG content was quantified using the standard dimethylmethylene blue (DMMB) assay using VersaMax UV-Vis spectrophotometer (Molecular Devices) (57), and DNA content was quantified using QuantiiT PicoGreen dsdna Reagent and Kits (Molecular Probes) per manufacturer s protocol using a VICTOR X3 Multilabel Plate Reader (Perkin Elmer). The GAG/DNA ratios were calculated from the results of these two assays Gene expression analyses Three hmsc pellets per group at 3, 7, 14, and 21 days of chondrogenic culture were washed with PBS and frozen at -80 C until gene expression analyses. Total RNA was extracted from each frozen pellets using TRIzol (Invitrogen). Briefly, pellets in 0.3 ml TRIzol were grinded using microtube pestles (USA Scientific), and the ground pellet samples were homogenized using QIAshredder columns (Qiagen). The lysate was transferred to a new 1.5 ml tube and incubated at room temperature for 5 minutes, after which the contents were centrifuged at 12,000 x g for 10 min at 4 C. RNease Mini Kit (Qiagen) was used for the rest of the RNA isolating process per manufacturer s protocol. The pre-designed human aggrecan, collagen-ii, collagen-i, collagen-x, 55

75 mmp-13 and 18S TaqMan primers were purchased from Applied Biosystems. Custom human sox-9 primers and probe (108) and human transforming growth factor-beta receptor II primers (forward: TCCACGTGTGCCAACAACATCAAC; reverse: TGCCACTGTCTCAAACTGCTCTGA) and FAM-labeled probe (ACAACACAGAGCTGCTGCCCATTG) were purchased from Integrated DNA Technologies. qrt-pcr reactions were done in duplicate in 384 well plates in a total volume of 10 μl as previously described (108) using 2x real-time TaqMan PCR Master Mix with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Relative expression levels normalized to 18S were calculated using the 2 -ΔCt method In vivo studies Animal surgeries All longitudinal animal experiments were performed following a University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) approved protocol (Protocol No ) using the previously established OCD model (42, 140). Briefly, OCD creation on twenty-four three-month old Sprague Dawley rats (Harlan) was performed under general gaseous anesthesia (Isoflurane, Phoenix Pharmaceuticals). The knees were shaved and swabbed with betadine and ethanol. A medial parapatellar approach was utilized to access the stifle joint. The patella was dislocated laterally and the knee was flexed to greater than 90-degrees to deliver the femoral trochlea into operative view. The trochlear groove was identified and the midpoint of the trochlea, which was the midpoint in the superior to inferior dimension at the center of the trochlear groove, was marked. A 1.5-mm drill was used to create the OCD, at a depth of 1.0-mm. 56

76 The subchondral bone was penetrated until bleeding bone was identified. Sterile saline was used to gently irrigate the area to remove any osteochondral fragments, and the incision was closed in layers. The animals were placed in standard cages with full access to food and water postoperatively. Subcutaneous buprenorphine (Reckitt Benckiser Healthcare) at 0.5 mg/kg dosage was injected twice daily for the first two-days following surgery as post-operative analgesia. Most of the study animals survived the OCD surgeries, except for one death associated with anesthesia complication. Hence, the number of animals per group in the in vivo study were N = 11 for No Doxycycline Control group and N = 12 for 12 Week Doxycycline group. The animals received either regular drinking water or 2 mg/ml doxycycline (Sigma-Aldrich) in the drinking water. Doxycycline water was replaced daily to ensure fresh supply of doxycycline. Table 4-1: ICRS Cartilage Repair Scoring System Criteria Points Scores In level with surrounding cartilage 4 75% repair of defect depth 3 Degree of defect repair 50% repair of defect depth 2 25% repair of defect depth 1 0% repair of defect depth 0 Complete integration with surrounding cartilage 4 Demarcating border < 1mm 3 ¾ of repair tissue integrated, ¼ with a notable border > 1 mm 2 Integration to border zone ½ of repair tissue integrated with surrounding cartilage, ½ with a notable 1 border > 1 mm From no contact to ¼ of repair tissue integrated with surrounding cartilage 0 Intact smooth surface 4 Fibrillated surface 3 Macroscopic appearance Small, scattered fissures or cracks 2 Several, small, or few but large fissures 1 Total degeneration of defect area 0 57

77 Repair tissue assessments Following sacrifice by carbon dioxide asphyxiation at 12 weeks post-ocd surgery, the joints were dissected and the repair tissue was examined grossly using three-dimensional (3D) optical coherence tomography (OCT) scanning system with a 1,310 nm center wavelength (Bioptigen) as previously described (141). The specimens were also analyzed with the aforementioned stereomicroscopy and camera, with/without india ink staining for gross grading using the ICRS- Cartilage Repair Assessment system (Table 4-1) (142). The harvested femurs were then fixed, decalcified, embedded in paraffin, and subsequently sectioned. The sections were stained using traditional hematoxylin and eosin (H&E) staining, as well as immunostained using rabbit polyclonal antibody against MMP-13. The stained sections were imaged using DP71 camera and Eclipse TE2000-U microscope, and evaluated using modified Holland histological scoring system (Table 4-2) (42). Three independent blinded individuals graded both the gross and sectioned histologic specimens, and the scores were averaged for statistical analyses. For the estimate percentages of MMP-13-positive cells, the sections from four selected knees from each treatment groups were imaged at high-power fields (magnification, x 20). Positive and negative cells were counted using ImageJ software (NIH), and percentages were calculated by dividing the numbers of MMP-13-stained cells by the sum of the numbers of MMP-13-stained and unstained cells. On average, 304 cells were counted per OCD repair tissue. The MMP-13 IHC images in Fig. 4-6C had the contrast increased by the same value in Adobe Photoshop CS4 to improve MMP-13-positive and -negative cell contrast for print and cell counting. 58

79 of less than 0.05 were considered significant, unless otherwise noted. In vitro studies were performed in triplicate (n = 3), and data are reported as line graphs with mean ± standard error of mean (SEM) (Figs ). In vivo studies were conducted and data observed using independent animals, with N = 11 for No Doxycycline Control group and N = 12 for 12 Week Doxycycline group, and data are reported as bar graphs with mean ± SEM for parametric data (Fig. 4-5) and as a boxplot with median and interquartile range for non-parametric data (Fig. 4-6B). For comparison among three groups and multiple time points for the in vitro data, two-way independent analysis of variance (ANOVA) was used with doxycycline concentration and time points as main independent variables. For variables that were considered significant, post-hoc Tukey was used to further evaluate the data. For comparisons between two groups for the in vivo data, unpaired Student s t-test was used for parametric data (Figs. 4-5C) and Mann-Whitney U test was used for non-parametric data (Fig. 4-6B). 60

80 4.3 RESULTS Figure 4-1: Macroscopic appearance of hmsc pellets and pellet area analysis (A) Macroscopic images of hmsc pellets cultured in chondrogenic media with 0, 1, or 2 μg/ml doxycycline (Dox) for 14 and 21 days. (B) Pellet area measured from the macroscopic images of hmsc pellets cultured in chondrogenic media with 0, 1, or 2 μg/ml Dox for 14 and 21 days. Pellet area increased from 14 to 21 days in chondrogenic culture period (p = 0.002). As well, the pellet area was significantly affected by the doxycycline concentration in the chondrogenic media (p = 0.002): 0 μg/ml Dox pellets were smaller than the 1 μg/ml (p = 0.014) and 2 μg/ml (p = 0.002) Dox pellets. There was no difference in sizes between the 1 and 2 μg/ml Dox pellets (p = 0.535). n = 3/group Gross evaluation of hmsc chondrogenic pellets (Fig. 4-1) Gross images of the hmsc chondrogenic pellets were taken at 14 and 21 days of culture in chondrogenic media supplemented with 0, 1, or 2 μg/ml of doxycycline (Fig. 4-1A). When the pellet areas were measured, an increasing trend in pellet areas from 14 to 21 days in chondrogenic culture was seen (Fig. 4-1B). The areas of hmsc chondrogenic pellets were 61

81 significantly affected by both the concentration of doxycycline (p = 0.002) and the time in chondrogenic culture (p = 0.002). The post-hoc Tukey test revealed that the pellet areas were significantly larger for those cultured in 1 μg/ml (p = 0.014) and 2 μg/ml (p = 0.002) of doxycycline compared to those cultured without any addition of doxycycline. There was no significant difference in pellet areas between those cultured in 1 and 2 μg/ml doxycycline. The interaction effect between the two main variables, doxycycline concentration and culture time, was also non-significant (p = 0.377). Figure 4-2: Biochemical quantification of GAG and DNA in hmsc pellets (A) Total GAG content per hmsc pellets showed increasing GAG content with longer chondrogenic culture time (p < 0.001), with GAG content of the 7 day pellets significantly greater than those of the 3 day pellets (p < 0.001), those of 14 day pellets significantly greater than those of the 7 day pellets (p < 0.001), and those of 21 day pellets significantly greater than those of the 14 day pellet (p < 0.001). There was no significant effect of doxycycline (Dox) concentration on the total GAG content (p = 0.412). (B) Total DNA content per hmsc pellets showed significant effect of chondrogenic culture time on the DNA contents (p < 0.001): DNA content of the 3 day pellets were significantly lower than those of the 7 (p < 0.001), 14 (p < 0.001), and 21 (p = 0.009) day pellets. There was no difference in DNA content among 7, 14, and 21 day pellets. As well, there was no significant effect of doxycycline concentration on the DNA content (p = 0.314). (C) Normalized GAG content with respect to the DNA content showed that there is significant effect of chondrogenic culture time (p < 0.001); however, the effect of doxycycline concentration was not significant (p = 1.000). n = 3/group. 62

82 4.3.2 Biochemical analyses of hmsc chondrogenic pellets (Fig. 4-2) Total GAG (Fig. 4-2A) and DNA (Fig. 4-2B) content per pellet samples and their ratio (Fig. 4-2C) were measured and calculated on 3, 7, 14, and 21 day old chondrogenic pellets to analyze the effect of doxycycline on hmsc chondrogenesis and proliferation/viability. For all three outcome measures, there was a significant main effect of the time in chondrogenic culture (ps < 0.001); however, the main effect of doxycycline concentration and the interaction effects were non-significant. The post-hoc Tukey tests for total GAG content and GAG/DNA ratio revealed significant differences of these outcome variables between all the different time points (ps < 0.001). On the other hand, the post-hoc Tukey test for total DNA content showed significant differences between day 3 and day 7 (p < 0.001), day 14 (p < 0.001), and day 21 (p = 0.009), but no significant differences between any other days Gene expression analyses of hmsc chondrogenic pellets (Fig. 4-3) Various mrna levels were measured on 3, 7, 14, and 21 day old chondrogenic pellets to analyze the effect of doxycycline on hmsc chondrogenesis. The time in chondrogenic culture had significant effect on gene expressions for all the assayed genes: sox-9 (p = 0.001), tgf-βrii (p = 0.003), aggrecan (p < 0.001), collagen-ii (p < 0.001), collagen-i (p = 0.028), collagen-x (p < 0.001), and mmp-13 (p < 0.001). The main effect of doxycycline concentration as well as interaction effects between the two main independent variables were non-significant for sox-9, tgf-βrii, aggrecan, collagen-ii, collagen-i, and collagen-x mrna levels. For the mmp-13 gene expression, on the other hand, there was a non-significant main effect of doxycycline concentration but a significant interaction effect between the time in chondrogenic culture and 63

83 doxycycline concentration (p = 0.025). This indicates that the doxycycline concentration affected mmp-13 mrna level differently on various chondrogenic culture time points. Specifically, mmp-13 levels for all three doxycycline concentrations were similar for day 3, 7, and 14 hmsc pellets; however, they were different for day 21 pellets (p = 0.012). The 2 μg/ml doxycycline pellets had significantly lower mmp-13 mrna level compared to 0 μg/ml doxycycline pellets (p = 0.010). It was lower compared to 1 μg/ml doxycycline pellets as well, however not to a significant extent (p = 0.078). Figure 4-3: Gene expression analyses of hmsc pellets Messenger RNA levels of chondrogenic inducers sox-9 and tgf-βrii, chondrogenic genes aggrecan and col-ii, fibrocartilage marker col-i, and hypertrophic chondrocyte marker col-x, and catabolic gene mmp-13. There was an increasing trend in sox-9, tgf-βrii, aggrecan, col-ii, and col-x levels (ps < 0.01), and a decreasing trend in col-i level (p = 0.028) over chondrogenic culture time. There was no significant effect of doxycycline concentration on these gene expressions (ps > 0.05). For mmp-13 level, there was an upward trend until day 14, with mrna level in the 14 day pellets significantly higher than those of the 3 (p < 0.001) and 7 (p < 0.001) days. However, on day 21, there was a reduction in the mrna level from day 14 (p < 0.001). The mmp-13 mrna levels on day 21 pellets were also significantly different among different doxycycline concentrations: 2 μg/ml doxycycline (Dox) pellets had significantly lower mmp-13 levels compared to 0 μg/ml Dox pellets (p = 0.01). n = 3/group. 64

84 4.3.4 Histological analyses of hmsc chondrogenic pellets (Fig. 4-4) Safranin O-fast green staining of sulfated GAGs showed increased amount of staining for 21 day versus 14 day hmsc pellets (Fig. 4-4A). However, no differences in staining intensities were appreciable among the three different doxycycline concentrations. These results confirm the biochemical analysis of total GAG content (Fig. 4-2A). The MMP-13 IHC showed more MMP- 13-positive cells for the 0 and 1 μg/ml doxycycline pellets at Day 21 (Fig. 4-4B), confirming the mrna results (Fig. 4-3). As upregulation of MMP-13 in MSC chondrogenesis has also been linked to terminal differentiation of hypertrophic chondrocytes ( ), alizarin red staining was performed. However, there were no positively stained pellets at either doxycycline concentration at both 14 and 21 days chondrogenic culture time points (Fig. 4-4C). Figure 4-4: Histological analyses of hmsc pellets Representative images of (A) safranin-o fast green, (B) MMP-13 immunohistochemistry, and (C) alizarin red staining of hmsc pellets at 20x (larger box) and at 4x (smaller inset). There was no difference in staining intensities for Safranin-O fast green and alizarin red staining among different doxycycline concentration groups at each time point. However, more MMP-13-positive cells were found in the periphery of the hmsc pellets cultured in 0 and 1 μg/ml doxycycline compared to those cultured in 2 μg/ml doxycycline. 65

86 Tomography (Fig. 4-5A). The animals that received doxycycline for 12 weeks post-op had higher mean total ICRS Cartilage Repair Scores reflecting better quality repairs than the No Doxycycline Control group (Fig. 4-5C, p =.017). Qualitative evaluation using the modified Holland Histological scoring of the H&E stained OCD repair tissue showed similar scores between the two groups (p =.116). Figure 4-6: MMP-13 of osteochondral defect repair tissues (A) Representative 4x (larger box) and 20x (smaller inset) images of MMP-13 immunohistochemical staining of repair tissues from No Doxycycline Control and 12 Week Doxycycline groups (B) Median and interquartile range of percentage of MMP-13-positive cells in different treatment groups. More MMP-13-positive cells were found in the newly formed repair tissues of No Doxycycline Control group compared to those of 12 Week Doxycycline group (p = 0.029). N = 4/group MMP-13 of OCD repair tissue (Fig. 4-6) The repair tissues from No Doxycycline Control group showed more MMP-13-positive cells compared to those of 12 Week Doxycycline group (Fig 4-6A): No Doxycycline Control group 67

87 had 80% MMP-13-positive cells, whereas 12 Week Doxycycline group had 44% MMP-13- positive cells (Fig 4-6B, p =.029). This in vivo OCD repair tissue MMP-13 content result correlates to the in vitro hmsc pellet data (Fig. 4-4B). 4.4 DISCUSSION Osteoarthritis currently affects 27 million adults and costs 3% of the GDP in the US (5, 146). The prevalence of osteoarthritis will continue to rise, especially with the aging population and the growing epidemic of obesity. By 2030 it is projected to affect 67 million, with 3.5 million total knee arthroplasties performed annually (5, 146). Therefore there is significant clinical as well as research interest in finding novel ways to improve the quality of cartilage repair. Articular cartilage repair with MSCs is widely studied; however, results are often suboptimal to prevent osteoarthritis. Oral administration of doxycycline has been postulated to have beneficial effects on articular cartilage by inhibiting important classes of MMPs (132, 147). Doxycycline administration in vivo has resulted in significant decreases in MMP levels in small animal models, as well as in human osteoarthritic patients (148, 149). When doxycycline was administrated prophylactically in several animal models, the decreased activities of MMPs were correlated to reduced proteoglycan loss from cartilage extracellular matrix, as well as reduced degenerative changes in the weight-bearing areas of cartilage (126, 150). In a randomized, double-blinded, placebo-controlled study that evaluated the effects of doxycycline on the progression of joint space narrowing in mildly arthritic knees, the doxycycline treatment group had a decreased rate of narrowing by 40% at 16-months and 33% at 30-months as compared to 68

88 placebo (p =.017) (133). On the other hand, however, a number of other studies show lack of any effect on joint disease and/or MMP activities with doxycycline treatment. In a prospective clinical study involving dogs with stifle joint instability, there was no significant difference in MMP activity levels in synovial fluid for doxycycline versus no doxycycline treatment groups (144). In another study involving guinea pigs, oral doxycycline treatment for 4 8 months lead to no significant OA changes compared to controls (151). Hence there are numerous studies on the effect of doxycycline on cartilage degeneration, with conflicting results. Nonetheless, doxycycline effect on cartilage repair with MSCs as well as MSC chondrogenesis is largely unknown. Our laboratory has also previously utilized doxycycline as a control molecule for external control of adeno-associated virus (AAV)-mediated transgene expression, as potential strategy to deliver therapeutic proteins of interest to affected joints to enhance MSC chondrogenesis, thereby improving cartilage repair (47). Intra-articular injection of AAV has been used in clinical trials for delivery of bioactive substances (88), and our previous study has shown both stable and persistent reporter transgene expression at one year after a single intra-articular injection of AAV (47). Nonetheless, there are potential safety issues concerning long-term or high-dose expression of transgenes, such as overexposure resulting in undesirable side effects. For this reason, controllable vectors have been developed to limit treatment time/dosage to the minimal needed to achieve therapeutic efficacy. One novel system is the Tet-on promoter, in which control is facilitated by using tetracycline-class drugs as activators of the tetracycline response element (TRE)-promoter, stimulating transgene expression only in the presence of the drug. Previous studies in our laboratory have successfully modulated gene expression utilizing the Tet-on 69

89 system with oral administration of 2 mg/ml doxycycline (47), providing an added measure of safety, as well as the ability to alter the timing and duration of exposure to bioactive factors. Our comprehensive in vitro study shows that doxycycline reduced the MMP-13 gene as well as protein expressions at day 21 of chondrogenic culture period. This is consistent with MMP-13 suppressing activity of doxycycline found in other tissue types, such as chondrocytes and synoviocytes (134, 136). We also observed larger hmsc pellets in the doxycycline group. The larger hmsc pellet sizes may be in part due to improved matrix synthesis through inhibition of MMP-13 production. As the predominant function of MMP-13 in cartilaginous ECM is collagen-ii breakdown (134, 144), this MMP-13 inhibition would ultimately lead to reduction of excessive collagen-ii degradation or enhancement of collagen-ii accumulation. MMP-13 is also a marker of terminal differentiation of MSCs into hypertrophic chondrocytes ( ). However, lack of differences in col-x gene expression level and lack of alizarin-red positive staining among hmsc pellets cultured in various doxycycline concentrations suggest that doxycycline is not principally involved in inhibition of MSC hypertrophy. Our in vivo data additionally suggests that oral administration of doxycycline potentially improves the repair. As well, MMP-13 expression in OCD repair tissue samples was significantly greater in No Doxycycline Control than in 12 Week Doxycycline groups. This can potentially explain the higher quality repairs reflected in the ICRS scores in the doxycycline treated rats. Furthermore, this in vivo finding is correlated to the in vitro findings of MMP-13 levels in control versus doxycycline-treated MSC pellets. 70

90 4.5 CONCLUSIONS In summary, we showed in both in vitro and in vivo study that doxycycline inhibited MMP-13 and potentially improved in chondrogenesis and cartilage repair. There was significant inhibition of MMP-13 on both mrna and protein levels in hmsc cultured with 2-µg/mL doxycycline, which would lead to reduction of collagen-ii degradation and hence larger pellet size potentially due to enhanced collagen-ii retention. The doxycycline-mediated inhibition of MMP-13 expression in MSCs is consistent with other studies using doxycycline on other joint cell and tissue types. Oral administration of doxycycline visibly improved osteochondral defect repair tissues in vivo. The doxycycline-mediated downregulation of MMP-13 expression was observed in OCD repair tissues. These findings suggest that doxycycline can be used as an agent for external control of transgene expression in cartilage repair studies incorporating AAV-mediated gene therapy strategies without interfering with the repair process. Doxycycline inhibition of MMP-13 may even potentially improve in vivo cartilage repair. As such, continued evaluation of the potential benefits of doxycycline to articular cartilage may lead to novel treatment strategies improve cartilage repair and delay development of osteoarthritis. 71

91 5.0 RELEASE OF BIOACTIVE ADENO-ASSOCIATED VIRUS FROM FIBRIN SCAFFOLDS: EFFECTS OF FIBRIN GLUE CONCENTRATIONS 5.1 INTRODUCTION Articular cartilage has a limited self-regeneration capacity and recombinant adeno-associated virus (AAV) is a promising vehicle for articular cartilage and the surrounding soft tissues to deliver bioactive factors to promote joint restoration. There are three approaches for AAV administration to articular cartilage: direct, indirect, and hybrid. The direct method involves an intra-articular injection of viral vectors into the articular joint space for their transduction of local cells. Although long-term persistence and transgene expression has been observed with direct injection of AAV (47), most of the transduction occurs in soft tissues and it is difficult to localize the transduction to specific cell types. Further, rapid dispersion of viral particles from the joint space would prevent effective transduction of repair cells that are recruited to the defect site over time. Thus, the in vivo transduction efficiency remains low and non-specific transduction of adjacent tissues and their transgene expression at undesired sites is usually observed (53). The indirect approach involves implantation of cells that were genetically modified ex vivo, and faces high clinical translational barriers. The hybrid approach, involving implantation of bioscaffold embedded with genetic material in the site of defect, leads to direct and localized transduction of host cells in situ and sustained expression of the transgene. Further, scaffolds can act as support 72

92 for injury-activated repair or progenitor cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), to migrate, attach, and differentiate at the defect site. Thus, the hybrid method offers the advantages of site-specific localization, direct transduction of host repair cells, and sustained transgene expression, while limiting the disadvantages of indiscriminate transduction and requirement of ex vivo manipulations. Our group has previously shown that AAV2-transforming growth factor-β1 (AAV2- TGF-β 1 ) transduced human MSCs (hmscs) implanted into a 1.5-mm diameter osteochondral defect significantly improved cartilage regeneration over 12 weeks in vivo (42). These results demonstrated that AAV2 is a suitable vector for gene delivery to improve the cartilage repair potential of the MSCs. However, although the ex vivo gene transfer is effective, the regulatory barriers for using genetically modified cultured human cells for therapeutic purposes clinically are extremely high. In addition, extraction and expansion of MSCs prolong the time before treatment can be applied and are very expensive. Therefore, we have explored the use of biodegradable scaffolds for release and delivery of bioactive substances to host MSCs within cartilage wound (140, ). Fibrin glue (FG) is used in a variety of clinical applications and in the laboratory for localized and sustained release of factors potentially important for tissue engineering. It has been shown that diluted FG produces a more open fibrin network compared to undiluted FG scaffolds (155), and that FG can act as an efficient scaffold for gene delivery ( ). However, the effect of different fibrinogen concentrations on FG scaffold delivery of bioactive AAVs has not been established. In the current study, we investigated the effect of different fibrinogen dilutions during the preparation of FG scaffold on the delivery of AAV2 and their early effects on human BM-MSC 73

93 (hbm-msc) chondrogenesis in vitro. The aim of this study was to test the hypotheses that diluted FG scaffolds will release more viral particles, resulting in higher transduction efficiency, and increase the chondrogenic potential of transduced hbm-mscs. 5.2 METHODS Preparation of FG Chemicals were purchased from Sigma-Aldrich unless otherwise stated. Twenty-four well plates and 24-well plate inserts were purchased from BD biosciences. Commercially available Tisseel human fibrin sealant (Baxter) was used to construct FG hydrogels. Fibrinogen (100% of the original material concentration, lyophilized human fibrinogen reconstituted in aprotinin fibrinolysis inhibitor) was prepared by serial dilution with phosphate buffered saline (PBS, Gibco) into four different concentrations: 25%, 50%, 75%, and 100% fibrinogen. Different dilutions of fibrinogen were subsequently mixed with an equal volume (1:1 volume ratio) of thrombin solution provided in the Tisseel kit (human thrombin IU/mL and calcium chloride μmol/ml). The resultant FG constructs were named 25%, 50%, 75%, and 100% FG, according to the fibrinogen dilution before thrombin activation. 74

94 5.2.2 Characterization of different FG concentrations Clotting time 100 μl of different fibrinogen dilutions was mixed with 100 μl Thrombin-CaCl 2 in a 96 well plate to make 25%, 50%, 75%, and 100% FG scaffolds. Change in the turbidity of the FG scaffold solution was immediately measured by a VersaMax UV-Vis spectrophotometer (Molecular Devices) at 550 nm wavelength (159). The clotting time was defined as the time at which the maximum value was reached in the absorbance curve. Three replicates of each FG scaffold concentrations were examined in this study Scanning electron microscopy Three FG scaffolds constructed from 200 μl of different fibrinogen dilutions and 200 μl Thrombin-CaCl 2 were used for scanning electron microscopy (SEM) analysis. After fixation in 2.5% gluteraldehyde for 1 h and rinsing in PBS, FG scaffolds were dehydrated in increasing alcohol concentrations (30%, 50%, 70%, 90%, and 100%) for 15 min each, followed by hexamethyldisilazane for 1 h. Once hexamethyldisilazane was removed, the scaffolds were airdried for an hour. Each FG scaffold was coated with an ultra-thin gold layer containing a gold sputter coater for 2 min at 25 ma and observed under an SEM (JSM-6335F; Jeol USA, Inc). Two separate images of each scaffold were captured. Pore size diameter and thickness of fibrin fibers were measured for each image using image analysis software, Metamorph (Molecular Devices). 75

95 FG dissolution Wet weight measurement Different fibrinogen dilutions were mixed with an equal volume of thrombin-cacl 2 and injected into uncoated 24 well plate inserts with 8 μm pores at 200 μl FG/insert, and incubated at 37 C for 15 min to form hydrogels. The weight of each insert was measured before injection of the fibrinogen/thrombin solutions. Once the FG scaffolds had solidified, 500 μl PBS was added on top of each inserts, and the inserts were placed in a 24 well plate containing 1 ml PBS/well. After incubation at 37 C for 10 min, inserts were removed, PBS aspirated, surface PBS absorbed by a filter paper, and the initial weight of the hydrogels (W 0 ) was recorded. FG hydrogels were re-weighed (W t ) every 2 days to estimate the wet weight fraction, which as defined as (W 0 W t )/W 0. Five replicates of each FG concentration were examined in this study Protein dissolution The conditioned PBS from each well, in which the FG hydrogels were incubated, was also collected at the time of hydrogel wet weight measurement. The amount of the protein dissolved and released into the PBS was measured by UV-Vis spectroscopy (NanoDrop) at 278 nm, which measures the absorbance of the phenyl group of fibrinopeptide proteins. Fresh PBS was used as blank control. 76

96 5.2.3 AAV2-CMV-GFP in vitro transduction AAV2-CMV-GFP-loaded FG preparation Double-stranded AAV2-GFP with a cytomegalovirus promoter (dsaav2-egfp, hereunder referred to as AAV2-CMV-GFP) was prepared as previously described (120). The AAV2 particles were packaged and purified using the adenovirus-free, triple plasmid transfection method. The titer (vector genome per milliliter [vg/ml]) was determined by viral DNA dot blot method (101). The stock AAV2-CMV-GFP titer was 3 x vg/ml. The AAV2-CMV-GFP vector stock was added to the various fibrinogen preparations (25%, 50%, 75%, and 100%) to obtain a final concentration of 6.25 x vg/ml. After vortexing for 3 min, the AAV2-CMV-GFP-loaded fibrinogen solutions were mixed with an equal volume of thrombin-cacl 2 and injected into uncoated 24 well plate inserts with 8 μm pores, at 200 μl/well. After gelation at 37 C for 15 min, pre-warmed 500 μl Dulbecco s modified Eagle s medium (DMEM, Gibco)-1% penicillin-streptomycin (pen/strep, Gibco) was added to each insert. Three replicates were examined in this study. AAV2-CMV-GFP released from the AAV2-CMV-GFP loaded FGs and their direct transduction of mammalian cells were assessed over 21 days. HEK-293 cells (ATCC) were maintained in DMEM, 10% fetal bovine serum (Atlanta Biologicals), and 1% pen/strep at 37 C and 5% CO 2. Each day, ~ 2.4 x 10 6 cells were seeded onto 24 well plates at a seeding density of 100,000 cells/well and incubated for 24 h, after which the medium was changed to serum-free DMEM-1% pen/strep. Inserts containing AAV2-CMV-GFP-loaded FGs were placed into the wells of the 24 well plate seeded with HEK-293 cells and incubated for additional 24 h at 37 C. The medium was placed below and in the inserts to allow migration of AAV2-CMV-GFP from 77

97 the FG hydrogels to the seeded cells. Photomicrographs of GFP-expressing cells were taken daily using an Olympus DP71 camera and Nikon Eclipse TE2000-U microscope. All GFP images presented in Figure-5-4C F had the contrast increased by the same value in Adobe Photoshop CS4 to improve the GFP signal for print AAV2-CMV-GFP release from FG Each day, the conditioned serum free medium from each well was collected and preserved at - 80 C until analysis. The amount of AAV2-CMV-GFP released into the conditioned medium from AAV2-CMV-GFP-loaded scaffolds was quantified by quantitative polymerase chain reaction (qpcr). All PCRs containing 0.5 μl of conditioned medium in a total volume of 10 μl using a commercially available pre-prepared 2x Real-time TaqMan PCR Master Mix (Applied Biosystems). GFP-specific custom TaqMan forward (GTCCGCCCTGAGCAAAGA) and reverse (TCCAGCAGGACCATGTGATC) primers as well as FAM-labeled probes (CCCAACGAGAAGCG) were used. All qpcrs were performed with an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) according to the following program: 12 min at 95 C, followed by 40 cycles of 15 s at 95 C and 1 min at 60 C. Three replicates of each experimental group were examined in this study In vitro transduction of AAV2-CMV-GFP released from FG Transduction efficiency was quantified using flow cytometry and expressed as percentage of GFP-positive cells. Both AAV2-CMV-GFP-transduced and non-transduced control cells were trypsinized, washed, and resuspended in 400 μl of PBS with 1% bovine serum albumin and 2 % 78

98 paraformaldehyde (both from Fisher Scientific). Ten thousand events were collected using a BD Biosciences LSR II (BD Biosciences). The percentage of GFP-positive cells was determined by defining GFP-negative cells and gating out debris with Flow Jo software (Tree Star, Inc.). Three replicates of each experimental group were examined in this study AAV2-CMV-TGF-β 1 in vitro transduction AAV2-CMV-TGF-β 1 -loaded FG preparation Double-stranded AAV2-TGF-β 1 with a cytomegalovirus promoter (dsaav2-tgf-β 1, hereunder referred to as AAV2-CMV-TGF-β 1 ) was prepared and purified as described above for AAV2- CMV-GFP preparation. The stock AAV2-CMV-TGF-β 1 titer was 2.5 x vg/ml. The AAV2-CMV-GFP and AAV2-CMV-TGF-β 1 vector stocks were separately added to 50% and 100% fibrinogen preparations to obtain a final concentration of 1.25 x vg/ml. After vortexting for 3 min, the AAV2-loaded fibrinogen solutions were mixed with an equal volume of thrombin-cacl 2 and injected into uncoated 6 well plate inserts with 8 μm pores, at 1 ml/well. After gelation at 37 C for 15 min, pre-warmed 3 ml chondrogenic medium: High- Glucose DMEM (Gibco) supplemented with 1% pen/strep (Gibco), 10-7 M dexamethasone (Sigma-Aldrich), 50 μg/ml L-ascorbic acid-2-phosphate (Sigma-Aldrich), 40 μg/ml proline (MP Biomedicals), and 1% ITS+ Premix (BD) was added to each insert. Three replicates of 50% and 100% AAV2-CMV-TGF-β 1 -loaded FGs were examined in this study against the 50% and 100% AAV2-CMV-GFP-loaded FGs. AAV2-CMV-TGF-β 1 released from the AAV2-CMV-TGF-β 1 -loaded FGs and their direct transductions of hbm-mscs were assessed. The hbm-mscs were obtained, maintained, 79

99 and characterized as previously described (57, 160). About 4.8 x 10 6 cells were seeded onto 6 well plates at a seeding density of 200,000 cells per well and incubated for 24 h in their normal growth medium, after which the medium was changed to a chondrogenic medium. Inserts containing AAV2-CMV-GFP-loaded FGs and AAV2-CMV-TGF-β 1 -loaded FGs were placed into the wells of the 6 well plates seeded with hbm-mscs and incubated for 24 h. The medium was placed below and in the inserts to allow migration of AAV2s from the FG hydrogels to the seeded cells. After the removal of the inserts, the cells were incubated for an additional 48 h at 37 C. The conditioned serum free medium and hbm-mscs from each well were collected separately and preserved at -80 C until analysis In vitro transduction of AAV2-CMV-TGF-β 1 released from FG The level of TGF-β 1 expression from hbm-mscs was assessed by determining the level of TGF-β 1 in cell culture supernatant using a commercially available ELISA (R&D Systems, Inc) according to the manufacturer s description, and using a CCL-64 mink lung epithelial growth inhibition assay as previously described with slight modification (161, 162). Briefly, the CCL-64 cell line (ATCC, CRL-1573) was grown in DMEM, 10% FBS, and 1% pen/strep at 37 C in 5% CO 2. CCL-64 cells were plated at 5,000 cells per well in 96 well plates and cultured in 100 μl of normal growth media for 3 h. The medium was then changed to 100 μl of serum-free DMEM- 1% pen/strep supplemented with 10% of conditioned media from hbm-msc culture. The plates were incubated at 37 C in 5% CO 2 for 24 h, and then 20 μl reconstituted XTT reagent from In Vitro Toxicology Assay Kit (Sigma-Aldrich) was added to each well for 2 h incubation. The absorbance at 450 nm was measured using VersaMax UV-Vis spectrophotometer. 80

100 Cartilage-specific gene expression was measured from hbm-mscs by quantitative reverse transcription-polymerase chain reaction (qrt-pcr). Total RNA from the collected hbm-msc samples was extracted using the RNeasy Mini Kit (Qiagen). The pre-designed human aggrecan and 18S TaqMan primers were purchased from Applied Biosystems. Human sox-9 custom forward (TGACCTATCCAAGCGCATTACCCA) and reverse (ATCATCCTCCACGCTTGCTCTGAA) primers as well as FAM-labeled probed (AGGCCAACCTTGGCTAAATGGAGCA) were purchased from Integrated DNA Technologies (IDT). All samples contained 1 μl of extracted RNA in a total volume of 10 μl using a commercially available pre-prepared 2x Real-time TaqMan PCR Master Mix. All qrt-pcrs were performed with an ABI PRISM 7700 Sequence Detection System according to the following program: 12 min at 95 C, followed by 40 cycles of 15 s at 95 C and 1 min at 60 C. Relative expression levels normalized to 18S were calculated using the 2 -ΔCt method Statistical analysis Data are expressed as mean ± standard error of mean. Statistical analysis was performed using independent samples t-test for comparison between two samples (Fig. 5-5), and one-way independent analysis of variance or one-way repeated measures analysis of variance with post hoc Tukey for multiple comparisons (Figs ) using Statistical Package for Social Studies (SPSS) 17.0 for Windows (IBM). The significance level was set as p <

101 5.3 RESULTS Clotting time After activation of the different fibrinogen dilutions with thrombin-cacl 2, there was an immediate change in the turbidity of the solution. The clotting time, or the time of maximal turbidity, was dependent on the fibrinogen concentration (p < 0.001). Absorbance of the fibrin gel at 550 nm increased with time for all fibrinogen dilutions until it reached a plateau. The clotting time as a function of fibrinogen concentration is displayed in Figure 5-1. Clotting time of the 100% FG was significantly higher (p < 0.05) than the diluted (25%, 50%, and 75%) FG scaffolds. Although there was no significant difference in clotting time between the dilute FG scaffolds, there was an increasing trend in clotting time with increasing FG concentrations. Figure 5-1: Clotting time of FG hydrogels as a function of fibrinogen concentration Clotting time showed an increasing trend with increasing fibrin glue (FG) concentrations. Clotting time of 100% FG was statistically higher than diluted FGs. Bars represent mean ± standard error of mean; n = 3. *p < 0.05 compared with 100% FG. 82

102 5.3.2 SEM analysis of the FG hydrogel microstructure Figure 5-2: Scanning electron microscopy analyses of FG hydrogels (A-D) SEM photographs of freeze-dried 25%, 50%, 75%, and 100% FGs, respectively. (E) Pore size analysis of the FG cross-sections showed that the pore diameter of the 100% FG was significantly lower than those of the diluted FGs. (E) FG fiber thickness analysis showed an increasing trend with increasing concentration of FG. The fiber thickness of each group was statistically different from each other. Bars represent mean ± standard error of mean; n = 3. *p < 0.05, # p < SEM analysis of pore size diameter of the FG scaffolds showed that all four scaffolds had pore size diameters ranging 50 1,200 nm. However, there was a denser, heterogeneous distribution of pores in the 100% FG (Fig. 5-2D) compared with the diluted FG hydrogels, which displayed a more homogeneous and larger pore size diameters (Fig. 5-2A C). The pore size of the 100% FG was significantly smaller in diameter (p < 0.05) than those of the diluted FG scaffolds (Fig. 5-2E). The fiber thickness of the scaffolds, also assessed by SEM, increased with increasing FG concentrations (Fig. 5-2F). The increases in fiber thickness were significant between scaffolds with different fibrinogen concentrations (p < 0.001). 83

103 5.3.3 In vitro dissolution of the FG hydrogels Analysis of in vitro FG dissolution in PBS showed a biphasic pattern (Fig. 5-3A). Wet weight fraction result showed that the dissolution rate is significantly affected by different concentrations of FG scaffolds (p < 0.001): 25% and 50% fibrinogen concentrations displayed a significantly higher rate of dissolution than 75% and 100% FG scaffolds. The first spike in dissolution occurred at day 2 for the 25% and 50% FG scaffolds, where the hydrogels lost ~35% and 15% of their initial weight, respectively. This was followed by a second spike at day 8, when the hydrogels lost ~80% of their initial weight. For the 75% and 100% FG scaffolds, the two spikes in dissolution occurred later at days 6 and 12, with loss of 20% and 90% of the initial weight for the respective time points. The pattern of protein released, observed by UV-Vis spectroscopy (Fig. 5-3B), correlates well with the wet weight fraction finding, by being significantly affected by the percentage of fibrinogen (p < 0.001). As well, the biphasic pattern of absorbance peaks that reflects most weight dissolution into the conditioned PBS can be noted. Figure 5-3: In vitro FG dissolution (A) Wet weight fraction analysis and (B) absorbance at 278 nm, reflecting the amount of phenyl group of fibrinopeptide proteins dissolved in the conditioned PBS, showed a biphasic dissolution pattern for all FGs. Data markers represent mean ± standard error of mean; n = 5. *p < 0.05 between 25% and 50% FGs and 75% and 100% FGs. 84

104 5.3.4 Quantification of the amount of released AAV2-CMV-GFP from FG hydrogels The amount of AAV2-CMV-GFP released into the conditioned media from the AAV2-CMV- GFP-loaded FG scaffolds is significantly affected by different concentrations of FG scaffolds (p < 0.001), and also displays a biphasic pattern (Fig. 5-4A). The spikes in the amount of AAV2- CMV-GFP released into the media occurred at day 2 and 9 for 25% and 50% scaffolds. In contrast, the spikes in the amount of AAV2-CMV-GFP released into the media occurred at day 2 and 15 for 75% and 100% FG scaffolds. Approximation of the area under the curves in Figure 5-4A suggests that the vector genomes of AAV2-CMV-GFP released into the media from 75% and 100% FG scaffolds were lower than that of 25% and 50% FG scaffolds. Figure 5-4: In vitro characterization of AAV2-CMV-GFP-loaded FG scaffolds (A) Vector genomes (vg) of AAV2-CMV-GFP released into 1 ml of conditioned media and (B) percentage of GFP-positive HEK-293 cells show biphasic AAV2-CMV-GFP release and corresponding spikes in percentage of GFP-positive cells. Data markers represent mean ± standard error of mean; n =3. *p < 0.05 between 25% and 50% FGs and 75% and 100% FGs. (C-F) Fluorescent and bright-field photomicrographs of HEK-293 cells incubated with AAV2-CMV-GFP-loaded 25%, 50%, 75%, and 100% FGs at day 8 correlates with the quantitative findings in A and B. 85

105 5.3.5 Quantification of mammalian cell transduction efficiency of released AAV2- CMV-GFP The percentage of GFP-positive cells from transduction of AAV2-CMV-GFP released into the conditioned media from the AAV2-CMV-GFP-loaded FG scaffolds is also significantly affected by different concentrations of FG scaffolds (p < 0.001), and showed a biphasic pattern (Fig. 5-4B). This pattern is similar to those of the FG dissolution (Fig. 5-3) and AAV2-CMV-GFP release (Fig. 5-4A). FG scaffolds with 25% and 50% fibrinogen concentrations yielded significantly higher percentage of GFP-positive cells than the 75% and 100% FG scaffolds. There were spikes in the percentage of GFP-positive cells at days 1 and 8 for all FG scaffolds, which correlate with the biphasic release pattern of high amounts of AAV2-CMV-GFP at these time points (Fig. 5-4A). Although 75% and 100% FG scaffolds showed similar biphasic spikes in the percentage of GFP-positive cells, the response was significantly lower compared with 25% and 50% FG scaffolds. Photomicrographs of GFP-expressing cells taken daily confirmed quantitative findings by flow cytometry (Fig. 5-4C F) Quantification and functional analysis of TGF-β 1 expression from hbm-mscs transduced with released AAV2-CMV-TGF-β 1 The amount of TGF-β 1 synthesized by hbm-mscs and released into the conditioned medium is significantly affected by different concentrations of FG scaffolds (Fig. 5-5A, B). Cells incubated with 50% FG loaded with AAV2-CMV-TGF-β 1 made 2.5-fold more active TGF-β 1 than those incubated with 100% FG loaded with AAV2-CMV-TGF-β 1 (p = 0.017, Fig. 5-5A). As well, the conditioned medium from the hbm-msc culture that was incubated with the 50% FG loaded 86

107 5.4 DISCUSSION Tissue engineering scaffolds allow for the delivery of cells or diffusible factors that can act locally to promote tissue regeneration (163, 164). The combination of gene therapy and tissue engineering offers the potential to direct progenitor cells proliferation and differentiation into functional tissue replacement (53). We have previously shown that hmscs transduced with AAV-TGF-β 1 improved the in vivo cartilage repair when the ex vivo transduced cells were implanted into osteochondral defects of athymic rats (42). However, in an effort to move beyond ex vivo gene transfer, the current focus of the project is on in situ delivery of therapeutic transgenes, such as TGF-β 1, directly to host MSCs residing in cartilage wounds using biodegradable scaffolds. This strategy can be thought of as augmenting microfracture, which is an accepted human clinical standard for treatment of full-thickness cartilage defects (165). The goal of this strategy is to induce localized exogenous transgene expression in repair/progenitor cells infiltrating into the scaffold for sustained release of these factors at the site of injury. Strategies that combine scaffolds, drug delivery technology, and gene therapy have the potential to provide more effective tissue regeneration with significant improvements on safety and cost issues compared to currently available therapies. Although numerous novel synthetic and natural polymers have been investigated for cartilage tissue engineering studies (140, 153, 154), usage of an FDA-approved and autologous biomaterial as an AAV2-releasing scaffold can accelerate the transition of basic science research into human clinical studies. FG is a biomaterial widely used in clinical practice and tissue engineering applications. As well, dilute FG has been successfully used to treat cartilage wounds in a pilot human clinical study (152). However, studies on its use as a delivery scaffold for gene vectors are limited, and only adenovirus delivery has been investigated for delivery of viral 88

108 vectors (55). FG has been investigated as a delivery scaffold for controlled release of adenovirus in a rabbit ear ulcer model (156) and for perivenous adventitial gene transfer (158), and was shown to provide enhanced in vivo transgene expression over adenovirus transduction without FG scaffold. For cartilage tissue engineering purposes, FG scaffolds with non-viral copolymerprotected polyethylenimine-dna vectors achieved sustained release of the gene over 20 day period and successful in vitro transfection of human keratinocytes and rabbit articular chondrocytes (157). Nonetheless, FG formulation for AAV2 delivery has not been optimized. The fibrinogen/thrombin concentration in the FG preparation has been shown to affect the structural properties of the scaffold, and in turn influence the proliferation rate and morphology of hmscs (155). Therefore, alterations in FG scaffold microstructure can influence its function as a delivery scaffold for AAV2. The aim of the current study was to assess, in vitro, the structural characteristics of FG hydrogels containing varying fibrinogen concentrations, their AAV2- particle release kinetics, and subsequent transgene expression in target cells for articular cartilage tissue engineering. Undiluted 100% FG scaffold had longer gelation time compared with diluted scaffolds, which may be explained by data from SEM analysis, which showed that 100% FG scaffolds formed a denser scaffold with smaller pore sizes and thicker fibrin fibers than diluted FG scaffolds. In contrast, diluted FG scaffolds had a more open fibrin network, which was reflected by their shorter clotting times. Consistent with another finding (155), diluted FGs had a more homogenous structure than undiluted 100% scaffold. FG scaffolds dissolved over time in PBS, which was shown by the decrease in the wet weight over time and by the change in absorbance measurements of the phenyl group of the 89

109 fibrinopeptide over time. Dissolution of the FG scaffolds coincided with the amount of AAV2- CMV-GFP released into the conditioned media. In addition, both the dissolution of the scaffold and the amount of AAV2-CMV-GFP released into the media occurred in a biphasic burst fashion, consistent with a typical hydrogel bulk-degradation pattern. The burst release pattern of AAV2- CMV-GFP was similar to the percentage of GFP-positive HEK-293 cells. There were more GFPpositive cells when a greater amount of AAV2-CMV-GFP was released into the media. Both the amount of AAV2-CMV-GFP released and the number of GFP-positive cells is higher in wells with 25% and 50% FG scaffolds than in those with 75% and 100% FG scaffolds. In addition to dissolution pattern of the FG scaffold, the more open fibrin network of the 25% and 50% FG scaffolds may contribute to greater release of AAV2-CMV-GFP and the higher percentage of GFP-positive cells compared with 75% and 100% FG scaffolds. These in vitro studies showed that commercial strength 100% as well as 75% FGs are less efficient at releasing AAV2 particles, and that dilute 25% and 50% FGs are more effective. Viral particles released from dilute FGs retained the ability to transducer HEK-293 cells up to 17 days in vitro. In contrast to our findings with AAV2, a previous study investigating different concentrations of FG scaffold loaded with adenovirus had achieved optimal viral releasing properties with the higher fibrinogen concentration (166). However, this was observed with adenovirus vector and with a diluted thrombin concentration. Compared with adenovirus, AAV is much smaller and has the advantages of improved safety profile and longer transgene persistence (47). In addition, lower thrombin concentration decreases the rate of fibrin crosslinking (55). Since in vivo FG decomposition will be accelerated via plasmin-mediated fibrin degradation in addition to diffusion-mediated dissolution, FGs prepared with undiluted thrombin are more likely to persist and release AAV2 particles longer for animal or human applications. 90

110 Although many more dilutions of FG at smaller increments could be studied to further assess the AAV2 release kinetics, especially with lower concentrations of FG, the four dilutions were chosen to support the goal of evaluating FG concentrations potentially useful for in vivo clinical applications. As well, FG dilutions of 25% approach the lower limit to achieve a clinically functional scaffold structure (166). For this reason, 50% and 100% FGs were chosen for subsequent studies with AAV2-CMV-TGF-β 1. As our ultimate goal is to develop a localized AAV2 delivery system for cartilage tissue engineering, we further explored the concentrations of 50% and 100% FGs for delivery of AAV2 encoding for a therapeutic gene useful for cartilage regeneration. Transforming growth factor-β 1 (TGF-β 1 ) has long been shown to consistently induce BM-MSC chondrogenesis in vitro (57, 167). We also have previously shown that implantation of AAV2-CMV-TGF-β 1 -transduced hmscs into osteochondral defects has improved cartilage repair in vivo (42). Therefore, we compared AAV2-CMV-TGF-β 1 delivery from the 50% and 100% FGs to hbm-mscs in vitro. The studies on FG scaffold delivery of therapeutic AAVs versus reporter gene AAVs, especially for their chondrogenic effects, have been limited to date (166). hbm-mscs cultured with 50% FG-AAV2-CMV-TGF-β 1 construct had higher concentration of active TGF-β 1, which resulted in greater inhibition of CCL-64 cell proliferation, and higher cartilage-specific gene expression levels in tranduced hbm-mscs. 5.5 CONCLUSIONS In summary, we have demonstrated that varying the fibrinogen concentrations in FG constructs changes the structural and functional characteristics of the scaffold for gene delivery. Diluting 91

111 the fibrinogen concentration yields a scaffold with a network of larger pores and thinner fibers, which reduces entrapment and subsequently enhances the release of AAV2 particles. Lower concentration FG scaffolds have also been shown to promote greater proliferation of hmscs, which are the principal cell types recruited for in situ chondrogenesis (155). Therefore, implantation of diluted FG scaffolds containing bioactive AAV2 vectors have the potential to improve articular cartilage regeneration and be translated for clinical cartilage tissue engineering. Further in vivo studies are planned using small and large animal models to validate the enhanced repair potential of diluted FG scaffolds with therapeutic AAV2-TGF-β 1 for localized in situ cartilage tissue engineering before translation into the clinical setting. 92

112 6.0 INTERLEUKIN-1BETA-MEDIATED INHIBITION OF HUMAN MESENCHYMAL STEM CELL CHONDROGENESIS IS BLUNTED BY ADENO- ASSOCIATED VIRUS GENE TRANSFER OF INTERLEUKIN-1 RECEPTOR ANTAGONIST AND TRANSFORMING GROWTH FACTOR-BETA1 6.1 INTRODUCTION Articular cartilage plays the essential role of forming a smooth surface between two bones, permitting frictionless joint motion. It is mainly composed of chondrocytes, as well as collagen type II and aggrecan that form a dense and avascular extracellular matrix (ECM). Due to the dense and avascular nature of ECM, the healing capacity of articular cartilage once damaged, is severely limited. Cartilage injuries, from trauma or degeneration, lead to debilitating osteoarthritis, often regardless of treatment status. Microfracture is a commonly used surgical technique to promote cartilage repair (168). This technique involves perforation of the subchondral bone to allow bone marrow contents to access and fill the defect and undergo chondrogenic differentiation. The bone marrow contents include bone marrow-derived mesenchymal stem cells (MSCs) and various growth factors. Numerous studies have shown successful MSC differentiation into the chondrogenic phenotype using various growth factors in a controlled in vitro environment with pre-defined chondrogenic differentiation media (61). The microfracture technique resulted in improved cartilage repair in 93

113 in vivo studies as well; however, the remodeled cartilage remains inferior to the native articular cartilage in terms of matrix composition and biomechanical quality (169). Microfracture, nonetheless, remains popular due to its convenience and minimally invasive nature (168). The altered homeostatic environment of the diseased joint may be responsible for the inconsistency between the in vitro and in vivo results of MSC chondrogenesis (80). In contrast to a healthy joint or a pre-defined in vitro environment, catabolic/inflammatory mediators are upregulated in diseased joints or from the surgical intervention itself, further impairing the ability of cartilage repair (170, 171). Indeed, addition of inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, in pre-defined chondrogenic media inhibited chondrogenic differentiation of MSCs in a dose-dependent manner (71, 80). When the cytokine actions were blocked using antagonist molecules in vitro, the inhibitory effect on chondrogenesis was restored to a certain extent (80). The anti-inflammatory molecules used in this study include IL-1 receptor antagonist protein (IRAP) and transforming growth factor (TGF)-β 1. IRAP is a small kda secreted protein with no known agonist function (74). It competitively inhibits IL-1 signaling via competitive inhibition of IL-1 ligand and receptor interactions (73). TGF-β 1, on the other hand, is a well-established anabolic growth factor that induces MSC chondrogenesis, and is often included in chondrogenic differentiation media (42, 57). In addition to its anabolic functions, TGF-β 1 has also been shown to decrease the catabolic activity of inflammatory cytokines in vitro and in vivo (64). However, although these anti-inflammatory strategies are straightforward, the in vivo efficacy is limited by the poor pharmacokinetics of the small molecule and the need for sustained high concentrations. 94

114 To overcome this limitation, genetic modification of MSCs has been explored for sustained delivery of the bioactive factors. Numerous vector carriers, such as adenovirus, lentivirus, and retrovirus, have been studied for gene transfer; nonetheless, significant safety concerns associated with these viruses limit their application to clinical gene therapy (123). Adeno-associated virus (AAV), in contrast, is a non-pathogenic virus that is already used in numerous human clinical trials (47, 119). AAV transduction of MSCs has also been extensively studied by us and others (16, 42, 108). In the current study, we investigated the chondrogenic differentiation of AAV-transduced MSCs in the presence of inflammatory cytokines in vitro. IRAP and TGF-β 1 molecules were overexpressed in the MSCs via AAV-gene transfer. The aim of the study was to test the hypothesis that chondrogenic potential of MSCs will be enhanced by using AAV-encoding for anti-catabolic factors, such as IRAP and TGF-β METHOD hmsc cell culture Human MSCs were obtained, processed, and maintained as previously described (57). Briefly, femoral bone marrow reaming tissues were obtained according to an exempt IRB-approved protocol at the University of Pittsburgh for discarded tissue. The bone marrow was minced, washed in phosphate buffered saline (PBS, Gibco), and vortexed. Then the cell suspension was passed through a cell strainer, loaded onto Histopaque-1077 density gradient (Sigma-Aldrich), and centrifuged at 400 x g for 30 min at 4 C to fractionate. Mononucleated cells were collected 95

117 6.2.4 Longitudinal transgene expression The transgene expression from AAV-transduced hmscs over the chondrogenic culture period was assessed by detecting GFP expression by live fluorescence with a MVX-10 MacroView Systems stereomicroscopy equipped with DP71 camera (both from Olympus), and determining the level of hirap in conditioned media at different time points using a commercially available ELISA (R&D Systems) according to the manufacturer s description. The GFP images in Fig. 1A had the contrast increased by the same value in Adobe Photoshop CS4 to improve image contrast for print Gross and histological assessments of hmsc pellets At the end of 21 days of chondrogenic culture, hmsc pellets were grossly assessed with stereomicroscopy and DP71 camera. The pellet area was measured using ImageJ (NIH). The pellets were subsequently fixed and processed for histological analyses using Safranin O-Fast green staining for sulfated glycosaminoglycan (GAG) according to standard protocols Biochemical analyses of hmsc pellets Chondrogenic hmsc pellets were also collected for biochemical analyses. Each pellet was papain-digested overnight in a 60 C shaking water bath (200 RPM) in 0.25 ml papain buffer. The composition of papain buffer was the following: 50 mm sodium phosphate (Sigma-Aldrich), 5 mm Ethylenediaminetetraacetic acid (EDTA, Gibco), 5 mm cysteine HCl (Sigma-Aldrich), and 0.5 mg/ml papain (Sigma-Aldrich). The total GAG content from the digested pellets was 98

118 quantified with the dimethylmethylene blue (DMMB) assay using VersaMax UV-Vis spectrophotometer (Molecular Devices) as previously described (57). The DNA content was also quantified with Quant-iT PicoGreen dsdna Reagent and Kits (Molecular Probes) and a VICTOR X3 Multilabel Plate Reader (Perkin Elmer) per manufacturer s protocol. The GAG content of the pellets was normalized by dividing the DNA from the GAG contents Statistical analyses All the statistical data analyses were performed using Statistical Package for Social Studies (SPSS) 17.0 for Windows (IBM), with the significance level set as p <.05 unless otherwise noted. Shapiro-Wilk and Levene s tests were used to test the assumptions of parametric data. The longitudinal IRAP transgene expression levels in conditioned media were analyzed using twoway mixed analysis of variance (ANOVA), with time as repeated measure and cytokine as independent variables (Fig. 1B). Pellet sizes and GAG contents were analyzed using two-way independent ANOVA with the AAV constructs and cytokine as independent variables (Figs. 2B, 3B, and 4B) and post hoc Tukey when there was a significant effect of AAV construct on the outcome variables (Figs. 3B and 4B). Student s t-test was used when the interaction effects were significant from the two-way ANOVAs to analyze the effect of cytokine stimulation. 99

119 6.3 RESULTS Figure 6-1: Longitudinal transgene expression from AAV-transduced hmsc pellet culture (A) GFP-expression from AAV-GFP transduced hmscs at 7, 14, and 21 days of chondrogenic culture, with and without IL-1β. (B) hirap concentration detected from conditioned culture media from AAV-hIRAP transduced hmscs at various time points of chondrogenic culture with and without IL-1β. n = 2 3/group. *p <.007 between CTRL and IL-1β at each time points Longitudinal transgene expression Robust GFP transgene expression was observed in AAV-GFP transduced hmsc chondrogenic pellets throughout the 21 day culture period (Fig. 6-1A). The IL-1β stimulated AAV-GFP group pellets, while smaller in size, showed brighter GFP signal than the control AAV-GFP group 100

120 pellets at all time points. No GFP-signal was observed from the untransduced control and AAVhIRAP or AAV-TGF-β 1 transduced pellets (Data not shown). Detectable levels of hirap transgene product were seen in AAV-hIRAP transduced hmsc chondrogenic pellets throughout the 21 day culture period (Fig. 6-1B). There was a significant main effect of time on the hirap concentration (p <.001), with gradual decrease in the transgene expression over time. Although there was no significant effect of cytokine stimulation (p =.276), the interaction effect between the time and cytokine stimulation was significant (p =.010). Hence, the cytokine effect was further explored. There was a significant effect of cytokine stimulation at days 14, 17, and 21 on hirap production (ps <.007): hmsc pellets cultured in the presence of IL-1β produced more hirap than those cultured without IL-1β. No hirap was detected from the untransduced control and AAV-GFP or AAV-TGF-β 1 transduced pellets (Data not shown). Figure 6-2: Macroscopic analysis of AAV-hIRAP-transduced hmscs at 21 days of chondrogenic culture (A) Representative images of hmsc pellets from untransduced, AAV-GFP, and AAV-hIRAP groups with and without IL-1β. (B) Area analysis of the pellets hmsc pellets from untransduced, AAV-GFP, and AAV-hIRAP groups with and without IL-1β. Mean fold pellet area was normalized against the area from untransduced no cytokine treated pellets. n = 2 3/group. *p <.05 between CTRL and IL-1β. 101

121 6.3.2 hmsc pellet area evaluation Macroscopic images of the hmsc pellets were taken at chondrogenic culture day 21 (Figs. 6-2 and -3). For the AAV-hIRAP pellet area analysis (Fig. 6-2B), there was a significant main effect of cytokine (p <.001). There was no significant effect of AAV constructs (p =.158); however, the interaction effect between the AAV construct and cytokine stimulation was significant (p =.001). When the cytokine effect on each of the AAV groups was further explored, the areas of hmsc chondrogenic pellets were significantly decreased by IL-1β stimulation for untransduced control and AAV-GFP groups (p =.025 and p =.024, respectively). On the other hand, the effect of cytokine stimulation for AAV-hIRAP group was not significant. Figure 6-3: Macroscopic analysis of AAV-TGF-β 1 -transduced hmscs at 21 days of chondrogenic culture (A) Representative images of hmsc pellets from untransduced, AAV-GFP, and AAV-hIRAP groups with and without IL-1β. (B) Area analysis of the pellets hmsc pellets from untransduced, AAV-GFP, and AAV-hIRAP groups with and without IL-1β. Mean fold pellet area was normalized against the area from untransduced no cytokine treated pellets. n = 3 4/group. *p <.05 between CTRL and IL-1β. 102

122 For the AAV-TGF-β 1 transduced hmsc pellets (Fig. 6-3B), their areas were significantly affected by both the AAV constructs and cytokine stimulation (ps <.001). As well, their interaction effect was significant (p <.001). Post hoc Tukey analysis showed that all three AAV construct groups were significantly different from one another (ps <.001 for all comparisons). The cytokine effect was further explored as well: the areas of hmsc chondrogenic pellets were significantly decreased by IL-1β stimulation for the untransduced control and AAV-GFP groups (ps <.001), but not for the AAV-TGF-β 1 transduced hmsc pellets hmsc glycosaminoglycan content GAG contents of hmsc pellets were stained, measured and calculated after 21 days of chondrogenic culture (Fig. 6-4). Via Safranin-O Fast Green staining (Fig. 6-4A), the untransduced control hmsc pellets underwent the best chondrogenesis, while the untransduced IL-1β stimulated hmsc pellets performed the worst. Comparing the non-il-1β stimulated control groups, the AAV-GFP and AAV-hIRAP transductions reduced the chondrogenic potential of hmscs, with AAV-hIRAP having greater effect than the AAV-GFP. On the other hand, when the pellets were treated with IL-1β, AAV-hIRAP transduction rescued the IL-1βmediated inhibition of hmsc chondrogenesis, while AAV-GFP had no effect. When the GAG content was quantified via biochemical analyses (Fig. 6-4B), there were significant main effects of AAV constructs (p =.031) and cytokine stimulation (p <.001). As well, the interaction effect between the two main effects was significant (p =.001). Post hoc analysis of the three AAV constructs revealed that there was a significant difference between AAV-GFP and AAV-hIRAP pellets (p =.009), but no differences between any other pellets. When the cytokine effect for each AAV constructs was analyzed, the IL-1β stimulation 103

123 significantly decreased the GAG content for the untransduced control (p =.014) and AAV-GFP groups (p =.001), but not for the AAV-hIRAP groups. A Untransduced AAV-GFP AAV-hIRAP CTRL IL-1β B * * Figure 6-4: GAG content of AAV-hIRAP transduced hmsc pellets at 21 days of chondrogenic culture (A) Representative Safranin O-Fast green images of hmsc pellets from untransduced, AAV-GFP, and AAV-hIRAP groups with and without IL-1β. (B) GAG content analysis of the pellets hmsc pellets from untransduced, AAV- GFP, and AAV-hIRAP groups with and without IL-1β. Mean ratio GAG content was normalized against that of the untransduced no cytokine treated pellets. n = 2 3/group. *p <.05 between CTRL and IL-1β. 104